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Inhibitors of human indoleamine 2,3-dioxygenas Vottero, Eduardo Ricardo 2007

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INHIBITORS O F H U M A N INDOLEAMINE 2,3-DIOXYGENASE By Eduardo Ricardo Vottero B.Sc, The University of British Columbia, 2001 A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F PHILOSOPHY in T H E F A C U L T Y O F G R A D U A T E STUDIES (BIOCHEMISTRY AND M O L E C U L A R BIOLOGY) T H E UNIVERSITY O F BRITISH C O L U M B I A January 2007 © Eduardo Ricardo Vottero, 2007 I A B S T R A C T Indoleamine 2,3-dioxygenase (IDO) is a tryptophan degradation enzyme that is emerging as an important drug target. IDO is expressed by many human tumors and results in suppression of the T-cell based immune response. IDO has also been implicated in depression and in the formation of senile nuclear cataracts. Recognition of these vital roles played by IDO, has led to considerable interest and resulted in the development of potent and selective IDO inhibitors for use in research and as lead compounds for drug development. In the present work, a bacterial expression system for production of recombinant human IDO has been constructed, and the purified enzyme has been used to develop a high-throughput activity assay for IDO that has enabled assay-guided fractionation of extracts prepared from marine invertebrates by the Andersen group at this university. Screening of this library of -4500 extracts led to the identification of potent inhibitors from two marine organisms. The polyketides annulin C, annulin A and annulin B were identified as the most potent IDO inhibitors from the hydroid Garveia annulata, and three quinones, adociaquinones A and B and xestoquinone were identified as potent IDO inhibitors from a sponge of the genus Xestospongia. Structure analogues of these natural compounds that are commercially available have also been studied. Most of these inhibitors exhibit K\ values greater than any reported previously, none are substrate analogs, and those that have been evaluated exhibit uncompetitive inhibition with respect to tryptophan. u The expression of human IDO in a Saccharomyces cerevisiae tryptophan auxotroph restricts yeast growth in the presence of low tryptophan concentrations, and inhibition of this IDO activity can restore growth. This growth response was used as an assay for EDO activity in vivo to screen crude extracts and the compounds present in the NCI Diversity Set for IDO inhibitors. In this manner, NSC 401366 (imidodicarbonimidic diamide, A7-methyl-N'-9-phenanthrenyl-, monohydrochloride) was identified as a potent non-indolic IDO inhibitor (K\ = 1.5 ± 0.2 uM) that is competitive with respect to tryptophan. The yeast growth restoration assay is simple and inexpensive. It combines desirable attributes of cell- and target-based screens and is an attractive tool for chemical biology and drug screening. The evolutionary relationship of IDO to some gastropod myoglobins suggests that IDO may undergo autoxidation in vivo such that one or more currently unidentified electron donors are required to maintain IDO heme iron in the active, ferrous state. To evaluate this hypothesis, yeast knockout mutants were used in combination with the yeast growth assay for EDO activity in vivo to demonstrate a role for cytochrome b$ and cytochrome b$ reductase in maintaining IDO activity in vivo. The availability of high throughput assays for EDO activity both in vivo and in vitro permitted the design of a variety of informative experiments that identify a number of new directions for study in which physical (kinetic, thermodynamic and spectroscopic) methods can be employed to understand the structural and mechanistic basis for the catalytic and physiological activities of this enzyme. iii T A B L E OF CONTENTS S E C T I O N P A G E ABSTRACT i i TABLE OF CONTENTS iv LIST OF FIGURES v i i LIST OF SYMBOLS, NOMENCLATURE AND ABBREVIATIONS v i i i ACKNOWLEDGEMENTS x i i i CHAPTER I - INTRODUCTION 1 1.1 - Overview 1 1.2 - Comparison of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) 2 1.3 - Biological roles of IDO 9 1.4 - Purification and structure of IDO 12 1.4.1 - Purification of authentic IDO from various sources 12 1.4.2 - Cloning and expression of human IDO 13 1.4.3 - Sequence alignment with other proteins 15 1.4.4 - Crystallography 16 1.5 - Physical and spectroscopic studies of IDO 22 1.6 - Mechanistic studies of IDO 26 1.7-IDO inhibitors 29 iv 1.8- Uncatalyzed oxidation of tryptophan 34 1.10- Goals of the current study 36 CHAPTER II - MATERIAL AND METHODS 38 2.1 - Reagents 38 2.2 - Biological material 38 2.3 - Protein expression and purification of human IDO 38 2.4 - In vitro IDO activity: discontinuous method 44 2.5 - In vitro IDO activity: continuous method 44 2.6 - In vitro screening of natural product library 44 2.7 - Yeast strains and vectors 45 2.8 - GFP-IDO construct 48 2.9 - In vivo IDO activity assay based on yeast growth inhibition 49 2.10 - Libraries of random IDO variants 50 2.11- Cytochrome b5 and IDO interaction in yeast 51 2.12 - Construction of the His346Gly IDO variant... 52 2 .13- Preparation of yeast cell lysates 53 2.14- Western blots 54 CHAPTER III - RESULTS 55 3.1 - Characterization of recombinant IDO 55 v 3.2 - Detection and Identification of IDO inhibitors present in extracts of marine invertebrates 55 3.2.1 - Inhibitors from the pacific hydroid Garveia annulata 55 3.2.2 - Inhibitors from sponges of the genus Xestopongia 58 3.2.3 - Structural analogues of IDO inhibitors obtained from marine invertebrates 64 3.3 - Inhibition kinetics of 1 methyl-1,4-naphthoquinone 64 3.4 - Development of an assay for IDO activity in vivo 66 3.5 - Screening in vivo for inhibitors of IDO activity 71 3.6 - Effects of naphthoquinones on yeast cells expressing human IDO 76 3.7 - Cytochrome b5 and IDO 79 3.8 - Random mutagenesis of IDO 85 CHAPTER IV - DISCUSSION 90 4.1 - Overview 93 4.2. - Novel IDO inhibitors families 93 4.2.1 - G. annulate family 93 4.2.2 - Xestoquinones family 93 4.2.3 - Naphthoquinones family 95 4.2.4 - NSC 401366 family 97 4.2.5 - Trisulfate polyhydroxysteroids family 98 4.3 - The phamacophore of human IDO 99 v i 4.4 - Inhibition mechanism of menadione 101 4.5 - IDO variants lacking activity in vivo 104 4.6 - Strategies for evaluation of human enzyme function in vivo with yeast 106 4.7 - Cytochrome b$ and IDO interaction in yeast 107 4.8 - Possible role of the residues 135,136 and 137 in IDO 113 4.9 - Proposed molecular interaction between IDO and cytochrome bs 115 4 . 1 0 - Future directions and concluding remarks 116 R E F E R E N C E S 122 v i i LIST OF FIGURES FIGURE TITLE PAGE Figure 1 - Structure of protoheme IX (iron protoporphirin IX, heme b).. 3 Figure 2 - The mammalian kynurenine pathway of tryptophan degradation 5 Figure 3 - Sequence alignment of IDO-like myoglobins and mammalian IDOs... 18 Figure 4 - Phylogenetic tree based on the alignment in Figure 3 19 Figure 5 - Structure of IDO-PI complex 20 Figure 6 - Active site of IDO-PI complex 21 Figure 7 - Proposed ionic reaction mechanism for conversion of tryptophan to N-formyl-kyrurenine catalyzed by indoleamine 2,3-dioxygenase 28 Figure 8 - Inhibitor 680C91 ((E)-6-fluoro-3-[2-(3-pyrildyl)vinyl]-1 H-Indole) of tryptophan 2,3-dioxygenase (TDO) 33 Figure 9 - Uncatalyzed oxidation of tryptophan 35 Figure 10 - Functional map of the 6.553 kbp plasmid pET 28 - IDO.... 40 Figure 11 - Sequence of the IDO cDNA. 43 Figure 12 - Chemical reactions related to the in vitro assay described in section 2.7 46 Figure 13 - Screen plate with inhibitors from the natural library 47 Figure 14 - Properties of recombinant human IDO 56 viii Figure 15 - Garveia annulata collected at Barclay Sound (British Columbia) 57 Figure 16 - Inhibition of IDO by 1-methyl-1,4-naphthoquinone 65 Figure 17 - Inhibition of yeast growth by human IDO 68 Figure 18 - Growth restoration as a drug-screening assay 70 Figure 19 - Compounds from the NCI diversity set that restore growth inhibited by IDO 72 Figure 20 - Growth restoration and direct inhibition of IDO activity by NSC401366 75 Figure 21 - Effect of vitamin K3, 1-methyl-1,4-naphthoquinone, on the growth level of human IDO expressing yeast 77 Figure 22 - Growth restoration of human IDO expressing yeast by several naphthoquinones 78 Figure 23 - Growth rate analysis of yeast cultures expressing wild-type IDO and His356Gly variant 81 Figure 24 - Growth rate inhibition cytochrome b5 deletion mutant 82 Figure 25 - Western Blot of the yeast lysates 83 Figure 26 - Growth rate inhibition of cytochrome b5 reductase deletion yeast mutant 84 Figure 27 - Growth rate analysis of yeast cultures expressing the T136K IDO variant 87 Figure 28 - Growth rate analysis of yeast cultures expressing KKK (wild type IDO), QQQ, QKQ, KQQ, QQK variants and empty vector 89 Figure 29 - Proposed pharmacophore structure for human IDO 100 Figure 30 - Proposed binding site for menadione at the active site of IDO as predicted by simulations with AutoDock 102 ix Figure 31 - IDO variants lacking activity in vivo 105 Figure 32 - Structure of cytochrome b5 catalytic fragment 108 Figure 33 - Location of Lys135,136 and 137 on the surface of IDO.... 114 Figure 34 - Stereo views of the proposed model for the interaction of IDO and cytochrome bsobtained by ClusPro 117 Figure 35 - Two different views between the proposed interaction between IDO lysyl residues and cytochrome glutamyl residues 118 x LIST OF TABLES T a b l e 2 - C o m p a r i s o n o f i n d o l e a m i n e 2 , 3 - d i o x y g e n a s e ( I D O ) a n d t r y p t o p h a n ( T D O ) T a b l e 3 - S u b s t r a t e s p e c i f i c i t y o f h u m a n i n d o l e a m i n e 2 , 3 -d i o x y g e n a s e T A B L E TITLE PAGE T a b l e 1 - H e m e d i o x y g e n a s e s 4 6 8 T a b l e 4 - P u r i f i c a t i o n o f H i s 6 - I D O f o r m 1 L c u l t u r e o f E. coli 1 4 T a b l e 5 - R e p r e s e n t a t i v e I D O i n h i b i t o r s 3 0 T a b l e 6 - C o m p o u n d s i d e n t i f i e d b y t h e in vitro a s s a y a s I D O i n h i b i t o r s 5 9 7 3 T a b l e 8 - M u t a t i o n s t h a t s u p p r e s s I D O a c t i v i t y in vivo 8 5 8 8 T a b l e 7 - E f f e c t o f N C I d i v e r s i t y s e t c o m p o u n d s o n I D O y e a s t g r o w t h r e s t o r a t i o n y e a s t a n d c a n c e r c e l l g r o w t h i n h i b i t i o n T a b l e 9 - I D O in vivo a n d in vitro a c t i v i t y f o r I D O v a r i a n t s s u b s t i t u t e d a t r e s i d u e s 1 3 5 - 1 3 7 T a b l e 1 0 - N o v e l I D O i n h i b i t o r f a m i l i e s i d e n t i f i e d b y s e v e r a l s c r e e n i n g Q . t e c h n i q u e s T a b l e 1 1 - C y t o c h r o m e 6 5 - d e p e n d e n t e n z y m e s y s t e m s w i t h N A D P H -c y t o c h r o m e P450 r e d u c t a s e o r N A D H - c y t o c h r o m e b5 1 1 1 r e d u c t a s e . T a b l e 1 2 - C y t o c h r o m e b 5 - d e p e n d e n t e n z y m e s y s t e m s w i t h N A D P H -c y t o c h r o m e P450 r e d u c t a s e 1 1 2 XI LIST OF SYMBOLS, NOMENCLATURE AND ABBREVIATIONS ABBREVIATION NAME 1MT 1-Methyl tryptophan CHES 2-(iV-cyclohexylamino) ethane sulfonic acid IDO Indoleamine 2,3-dioxygenase KO2 Potassium superoxide KPB Potassium phosphate buffer MTH-Trp Methyl-thiohydantoin-tryptophan O2 " Superoxide PI 4-Phenylimidazole T C A Trichloroacetic acid TDO Tryptophan 2,3-Dioxygenase Trp Tryptophan xn ACKNOWLEDGEMENTS I would like to thank all the past and present members of the Mauk laboratory because they instructed me in everything that I know about my profession. Along with the Mauk lab, this research was greatly aided by Aruna Balgi from the Roberge lab and by Alban Pereira from the Andersen Lab. Without their help, this thesis would not have been possible. I would specially like to thank my supervisor, Professor Grant Mauk, for his support and direction during my graduate studies and my advisory committee, Professor R.T.A. MacGillivray and Professor M . Roberge, for looking after me and for reviewing my thesis. Finally, I would like to thank my family for their understanding and support. Xll l CHAPTER I - INTRODUCTION 1.1 - Overview Prior to 1955, the sole metabolic role of dioxygen was believed to be that of the final electron acceptor in respiration. In that year, Mason demonstrated that an oxygen atom is incorporated directly from dioxygen into 3,4-dimethylphenol by phenolase (Mason, et al., 1955), and Hayaishi reported that pyrocatechuase transfers an oxygen atom from dioxygen into catechol (Hayaishi, et al, 1955). Hayaishi designated enzymes that incorporate oxygen atoms from dioxygen as "oxygenases." Since then, this general class of enzymes has been shown to play key metabolic roles in a wide range of organisms. Oxygenases are metalloenzymes and are classified into two groups based on the reaction that they catalyze: R-H + O2 + 2e" + 2H + —• R-OH + H 2 0 (1) R + 0 2 —• R(0) 2 (2) While reactions catalyzed by monooxygenases (Eq 1) result in incorporation of one oxygen atom of a dioxygen molecule into the substrate, reactions catalyzed by dioxygenases (Eq 2) result in the incorporation of both atoms of a dioxygen molecule. Most heme oxygenases are monooxygenases as represented by the large family of cytochromes P450. On the other hand, most dioxygenases are non-heme iron proteins, as 1 represented by pyrocatechuase. At present, only a few heme dioxygenases are known (Table 1), and these enzymes all possess iron protoporphyrin EX (heme b) (Figure 1). Although the detailed mechanistic role of the heme iron in heme-containing mono- and dioxygenases varies, in all cases the heme is required for activity, and catalytic intermediates in which oxygen interacts directly with the heme iron are believed to be critical to the reaction mechanism. Specific properties of each of these enzymes depend on (1) the identity of the proximal ligand, (2) the chemical environment provided by the heme binding site, and (3) the interaction of the substrate with the heme and its environment. In the 1950s and 1960s, two heme containing dioxygenases, tryptophan 2,3-dioxygenase (TDO) (Hayaishi, et al, 1957) and indoleamine 2,3-dioxygenase (IDO) (Yamamoto and Hayaishi, 1967) were reported to catalyze the same initial step of the conversion of L-Trp into Af-formyl kyrunenine by oxidizing the 2,3 double bond of the indole ring (Table 1). The original name for TDO was tryptophan pyrrolase. 1.2 - Comparison of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) Both IDO and TDO catalyze the first and rate-determining step of the kynurenine pathway in human metabolism (Figure 2). Although both enzymes are cytosolic heme proteins and catalyze the same reaction, only in the reduced (ferrous) forms do they exhibit the various properties summarized in Table 2. TDO has been isolated from Pseudomonas acidovorans and, in mammals, is expressed mainly in the liver (Maezono, etal., 1990). 2 COO' COO" Figure 1: Structure of protoheme IX (iron protoporphyrin IX, heme b) 3 Table 1: Heme dioxygenases Enzyme Reaction Catalyzed References Tryptophan 2,3-dioxygenase, Indoleamine 2,3-dioxygenase Prostaglandin H synthase Plant fatty acid cc-dioxygenases Linoleate diol synthase (fungal) tryptophan Arachidonic Acid COOH PGH2 Llnolenlc acid OOH COOH 2-Hydroperoxylinolenic acid Linolenlc acid .OOH COOH (Sono, etal, 1996) (Kulmacz and Lands, 1984; Lou , et al, 2000; Tsai, etal. , 1993) (De Leon, et al, 2002; L i u , et al, 2004) (Cristea, et al, 2003; O l i w and Su, 1997; Su, et al, 1998) 4 3-hydroxyl-L-kynurenine 2-amino-3-carboxy- 3-hydroxyl-anthranilate muconate semialdehyde quinolinate nicotinate nucleotide Figure 2 : The mammalian kynurenine pathway of tryptophan degradation. E n z y m e s a r e s h o w n i n r e d , a n d i n t e r m e d i a t e s a r e i n d i c a t e d i n b l u e . 5 Table 2: Comparison of indoleamine dioxygenase (IDO) and tryptophan dioxygenase (TDO) Properties IDOa TDO b Molecular weight (Kda) 45 191 (liver), 122 (Pseudomonas) Subunits 1 4 (ou0 or a2|32) Prosthetic group protoheme I X protoheme DC Oxygen source 0 2 (and 02*~) o 2 Substrates L- and D-Trp, 5-hydroxy-L- and D-Trp, tryptamine, serotonin L-Trp (and D-Trp)d ) Turnover no. for L-Trp (s 1) 2 7 (liver), 17 (Pseudomonas)^ Distribution Mammals Ubiquitous (except liver) Liver Bacteria None Pseudomonas acidovorans Inducers of gene expression mfluenza/HIV virus, lipopolysuccharide, interferon tryptophan, kynurenine, hydrocortisone (a) (Hayaishi, 1976) (b) (Sono, 1989) (c) (Maezono, et al, 1990) (d) (Watanabe, et al, 1980) (e) and (f) (Bianchi, et al, 1988) 6 On the other hand, IDO is expressed ubiquitously in mammalian tissues (except the liver) and has not been found in bacteria. IDO is a monomelic enzyme (45 kDa) whereas T D O is a tetramer (monomer M W 80 kDa) (Maezono, et al., 1990). The two proteins are antigenically distinct, and they do not share significant amino acid sequence homology. T D O is highly specific for oxidation of L-Trp, while IDO can oxidize a number of indole-containing compounds that are not substrates for T D O . The most relevant IDO substrates are shown in Table 3. Surprisingly, IDO reacts with superoxide to yield the oxygenated enzyme (Fe2+02), which can also be obtained by the reaction of dioxygen to the ferrous enzyme. The oxygenated IDO complex can be represented as Fe3+02*~. This superoxide reactivity of EDO is similar to those of horseradish peroxidase and catalase, but different from those of hemoglobin, myoglobin, cytochrome P450, or T D O , which present very low reactivities with superoxide (Sono, et al., 1996). T D O is a "substrate-inducible" enzyme, and tryptophan inhibits T D O degradation and increases its half-life (Sono, et al, 1996). For this reason, T D O seems to be primarily involved in the regulation of the level of tryptophan derived from dietary sources. On the other hand, EDO is not a "substrate-inducible" enzyme, and its biological role is more complex. The level o f IDO expression has been linked to the activity of the immune system, mainly to the level o f interferon gamma, the major inducer for the immune inflammatory response. The relationship between IDO and the immune system is discussed in greater detail in section 1.4, and the inhibition characteristics of IDO and T D O w i l l be discussed in Section 1.7. 7 Table 3: Substrate specificity of human indoleamine 2,3 dioxygenase Substrate Activity3' Km mol of product/mol of enzyme/min u M L-Tryptophan 101 21 D-Tryptophan 294 14,300 3-Hydroxy-L tryptophan 5 400 3-Hydroxy-D tryptophan 0.16 N d Tryptamine 0.04 N d Serotonin < 0.01 N d a ) (Takikawa, etal., 1988). 8 1.3 - Biological roles of IDO IDO catalyzes the initial and rate-determining step o f tryptophan metabolism via the kynurenine pathway in non-hepatic tissues (Figure 2). In recent years, IDO has been shown to play an important immunosuppressive function (Grohmann, et al, 2003) that has a variety of consequences. Specifically T-lymphocytes must divide to be activated, and this process is particularly sensitive to the availability of tryptophan. Cells expressing IDO can deplete their microenvironment o f tryptophan such that T-lymphocytes arrest in G l and fail to proliferate (Hwu, et al, 2000; Munn, et al, 1999; Munn, et al, 2002). Additionally, tryptophan metabolites from the kynurenine pathway are efficient T-cell inhibitors (Frumento, et al, 2002; Terness, et al, 2002). This function is important for regulating responses to autoantigens and also for reducing proliferation of parasites, bacteria and viruses (Grohmann, et al., 2003). IDO is highly expressed in the placenta where it plays a significant role in maintaining maternal tolerance towards the antigenically foreign fetus during pregnancy (Munn, et al, 1998). The immunosuppressive function of IDO can be exploited by tumor cells to escape immune detection. Many human tumors express IDO even though their tissue of origin does not (Uyttenhove, et al, 2003). IDO is also expressed by antigen-presenting cells at the periphery of tumors (Uyttenhove, et al, 2003) and in the lymph nodes that drain tumors where appropriate T lymphocytes would otherwise be activated (Munn, et al, 2004). That this mechanism of immune suppression can contribute to tumor evasion of T lymphocyte-mediated destruction has been demonstrated using immunogenic P815 tumor cells, which are normally rejected in mice immunized with the major P I A tumor antigen. However, when P815 cells are experimentally induced to express IDO, they are 9 not rejected but form lethal tumors (Uyttenhove, et al., 2003). EDO expression is also a marker of poor prognosis in ovarian cancer (Okamoto, et al, 2005). Tryptophan - derived U V filters (kynurenine, 3-hydroxylkynurenine and 3-hydroxyl-kynurenine glucoside) are synthesized by the kynurenine pathway in the human lens (Takikawa, et al, 2001). These U V filter compounds increase in amount with age, and they have been shown to bind to human lens proteins to produce yellowing of the lens (Takikawa, et al., 2003). Senile nuclear cataract is associated with coloration and insolubilization of lens proteins and with extensive oxidation of cysteine and methionine residues of crystallins. Korlimbinis et al. (2006) recently demonstrated that 3-hydroxykynurenine catalyzes the oxidation of methionine residues in both cd3- and aA-crystallin in vitro. 3-hydroxy-kynurenine promotes the oxidation and modification of crystallins and may contribute to oxidative stress in the human lens (Korlimbinis, et al., 2006; Takikawa, et al., 1999; Takikawa, et al., 2003). Because EDO is the first and rate-limiting step in the kynurenine pathway, yellowing and modification of the aged lens proteins may be preventable by pharmaceutical inhibition of EDO activity. EDO has also been implicated in depression and other neurological and psychiatric diseases through interference with serotonin production or accumulation of quinolate and other neuroactive kynurenine metabolites (Myint and Kim, 2003; Wichers and Maes, 2004). For example, Sardar et al (1995) indicated that HTV-associated dementia is a frequent consequence of HIV infection and relates to neuronal damage, possibly as a result of increased neurotoxic kynurenine metabolites. These authors measured EDO activity in post-mortem brain tissue from AEDS patients and observed that IDO activity was significantly increased in the samples from patients with dementia relative to those 10 without dementia. Thus, IDO activity was concluded to increase in HIV-associated dementia and to increase kynurenine pathway metabolites. Elevated levels of these neurotoxins may contribute to the neuronal deficits underlying HIV-associated dementia (Sardar and Reynolds, 1995). Using a model of HIV-1 encephalitis (HIVE), Potula et al (2005) investigated whether the EDO inhibitor 1-methyl-d-tryptophan could affect the generation of cytotoxic T lymphocytes (CTL) and clearance of virus-infected macrophages from the brain. Severe combined immunodeficient mice were reconstituted with human peripheral blood lymphocytes, and encephalitis was induced by intracranial injection of autologous HIV-1-infected monocyte-derived macrophages (MDMs). By week 3, the 1-methyltryptophan-treated mice exhibited an 89% reduction in HIV-infected MDMs in brain tissue as compared with controls. Thus, manipulation of immunosuppressive EDO inhibition by 1-methyl tryptophan in HIVE may enhance the generation of HIV-1-specific CTLs, leading to elimination of HIV-1-infected macrophages in brain (Potula, et al., 2005). Finally, a role for IDO in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (D3D). Using epithelial cells purified from patients, overexpression of indoleamine 2,3-dioxygenase was confirmed in Crohn's disease and in ulcerative colitis, as compared to normal mucosa. Potentially, this observation opens new avenues in the treatment of EBD (Barcelo-Batllori, et al., 2002). In short, IDO is an attractive target for therapeutic intervention in a number of major clinical applications. 11 1.4 - Purification and structure of IDO 1.4.1 - Purification of authentic IDO from various sources IDO was isolated first from rabbit small intestine (Yamamoto and Hayaishi, 1967), then from mouse intestine (Ozaki, et al., 1987) and finally from human placenta (Takikawa, et al, 1988). These different natural sources provided enzymes that had very similar enzymatic and physical chemical characteristics. Rabbit small intestine IDO was obtained by ammonium sulfate fractionation of intestinal homogenates from two rabbits (2 kg each) followed by chromatography in P-cellulose, DEAE-Sephadex A-50 to yield "purified" enzyme (4.8 mg) that represented 20% of the total initial IDO activity (Yamamoto and Hayaishi, 1967). Murine epididymal IDO was partially purified from a homogenate by ammonium sulfate fractionation followed by chromatography on Sephacryl S200 BioGel and by hydroxylapatite. The partially purified preparation was fractionated again by ammonium sulfate and the purified enzyme finally isolated by isoelectric focusing (Ozaki, et al., 1987; Ozaki, etal. 1986). Takikawa at al (1988) purified human IDO at least 10,000-fold from crude extracts of placentas following seven purification steps with a recovery of 0.9 %. In summary, five normal placentas (about 1.5 kg) were coarsely minced and rinsed with 0.9% NaCl to remove the blood. The purification steps included phospho-cellulose, hydroxyapatite, phospho-cellulose, Sephadex G-100, isoelectric focusing, C M affi-gel blue, and TSW C M 3SW chromatographic columns. From the crude extracts that 12 contained 182,900 mg of total protein, 0.17 mg of pure IDO was obtained (Takikawa, et al, 1988). 1.4.2 - Cloning and expression of human IDO The first recombinant bacterial expression system for IDO was developed by Littlejohn et al. (2000) and improved by Austin et al (2004) in Escherichia coli. A summary of purification of His6-EDO from the bacterial pellets of 1 liter of culture based on this technique is provided in Table 4. The enzyme was purified ~ 13-fold from the crude extract through two chromatography steps involving metal affinity chromatography (Ni-NTA agarose) and size exclusion chromatography (Superdex 75). The final sample gave a single band on SDS-PAGE. Recovery was 13 % from the crude extract and the yield was 3.5 mg/L of LB broth with an A s o r e t / A2so absorption ratio of 2.2 (Austin, et al, 2004). Recently, other E coli expression systems have been reported (Papadopoulou, et al, 2005; Sugimoto, et al, 2006). These expression systems follow the same procedures with minor modifications. As a result, only Sugimoto's technique will be described below. The coding region for human IDO was cloned into the pET-15b vector (Novagen) using the Ndel and BglE restriction sites. The plasmid encodes a His6-tag with a thrombin cleavage site in the A -^terminus of the full-length protein. The transformed E. coli BL21 (DE3) cells were grown in a rotary shaker at 30°C using LB medium containing ampicillin and 6-aminolevulinic acid. The expression of His-tagged IDO fusion protein was induced by the addition of isopropyl-D-thiogalactoside (IPTG) when the culture had grown to an OD600 of 1.0. Cell growth was continued for an additional 12 hours at 30 °C. 13 Table 4: Purification of His6-IDO from 1 L culture of transformed E. colt from Austin et al. (2004). Step Volume (mL) Total protein (mg) Total activity6 (umol/h) Specific activity (urnol/h/mg) Yield (%) Purification Crude extract 20.0 340 4216 12.4 100 1 N i 2 + - N T A agarose 3.50 10.6 1480 140 35 11.3 Superdex-75 0.25 3.5 560 160 13 13.0 a E. coli EC538 transformed with (pREP4, pQE9-JDO) b The enzyme activity was determined with L-tryptophan as the substrate 14 The harvested cells were resuspended in potassium phosphate buffer (pH 8.0, 200 mM NaCl) containing 2-mercaptoethanol (5 mM), imidazole (20 mM), PMSF (0.5 mM) and one tablet of complete (EDTA-free) protease-inhibitor cocktail (Roche). The suspension was homogenized with an ultrasonic processor after treatment with lysozyme for 30 min. The solution was centrifuged at 125,000 g for one hour, and cell debris was discarded. The supernatant fluid was then loaded onto a column of N i 2 + - N T A agarose resin. His-tagged IDO was then eluted with imidazole (150 mM). The eluted fraction was dialyzed against potassium phosphate buffer (50 mM, pH 8.0) containing NaCl (100 mM) and 2-mercaptoethanol (5 mM). The TV-terminal His tag was removed with the Thrombin Cleavage Kit (Novagen), and any uncleaved His-tagged IDO was removed by passing the solution through a column of N i 2 + - N T A agarose. The cleaved protein was dialyzed against Tris-HCl buffer (16 mM, pH 8.0) containing 2-mercaptoethanol (5 mM) and further purified by elution over a MonoQ column (Amersham). The protein was eluted with a linear gradient of 0-300 mM NaCl. Fractions with a high absorbance at 423 and 280 nm were pooled (Sugimoto, et al., 2006). 1.4.3 - Sequence alignment with other proteins In 1989, the myoglobin isolated from the abalone Sulculus diversicolor (Suzuki and Furukohri, 1989) was discovered to exhibit very low sequence similarity with other mammalian myoglobins; however, it shows 35% sequence identity with human IDO. To date, four IDO-like myoglobins have been identified from S. diversicolor (Suzuki and Furukohri, 1989), Nordotis madaka (Suzuki, 1994), Turbo cornutus (Suzuki, etal., 1998) and Omphalius pfeifferi (Kawamichi and Suzuki, 1998). The sequence alignments of 15 several EDO-like myoglobins, mammalian EDOs and a yeast hypothetical EDO are shown in Figure 3. This alignment provides evidence that mammalian IDOs and molluscan DDO-like myoglobins have evolved from a common ancestral gene as shown in the phylogenetic tree provided in Figure 4. 1.4.4 - Crystallography During the course of this work, the structure of recombinant human IDO was reported by Sugimoto et al (2006). From this analysis, the tertiary structure of IDO is found to be composed of a large domain, a small domain and an interconnecting loop (Figure 5). The large domain is comprised of 13 a-helices and two 3io helices. The small domain consists of six a-helices, two short p-sheets, and three 3io helices. The proximal ligand for the heme iron, His346, is located in helix Q (Figure 5). No distal residue is apparent in the crystal structure, contrary to the prediction based on EPR spectroscopy that IDO possesses a distal His residue (Sono and Dawson, 1984). The heme cavity is limited on one side by seven helices of the large domain. The top of the heme cavity is defined by the small domain and a long loop (residues 250-267) that connects the two domains. The heme cavity of the phenyl imidazole-EDO complex is shown in Figure 6. In the distal heme pocket of this crystal form, Sugimoto et al. (2006) observed clear density (Figure 5) for two molecules of 2-(N-cyclohexylamino) ethane sulfonic acid (CHES), a component of the crystallization buffer. The authors suggested that the high concentration of CHES results in binding of the buffer to the putative tryptophan-binding pocket. 16 O m p h a l i u s M b M P R L D V T Q F E V S M K T G F L I J 5 - N P L T K L P - A Y F D P W N N L A S T M A 4 1 t u r b o M b M P R I J J V A Q F D V S M K T G F I I ^ - N P L T K L P - A Y F D A W N N L S S K M S 4 1 S u l c u l u s M b M A D I Q L S K Y H V S K D I G F L L E - P L Q D V L P - D Y F A P W N R L A K S L P 4 1 N o r d o t i s M b M A D I Q L S K Y H V S K D I G F L L E - P L Q D V L P - D Y F E P W N R L A K S L P 4 1 m o u s e l D O M A L S K I S P T E G S R R I I i E D H H I D E D V G F A L P - H P L V E L P - D A Y S P W V L V A R N L P 5 1 r a t l D O M P H S Q I S P A E G S R R I L E E Y H I D E D V G F A L P - H P L E E L P - D T Y R P W I L V A R N L P 5 1 h u m a n l D O H A H A M E N S V T T I S K E Y H I D E E V G F A L P - O T Q E N L P - D F Y N D W M F I A K H L P 4 7 Y J R 0 7 8 W M N N T S I T G P Q V I j H R T K M R P L P V I j E K Y C I S P H H G F I ^ D R L P L T R L S S K K Y M K W E E I V A D L P 6 0 O m p h a l i u s M b Q H V A - N K T M R D E V A K L P V L D F N K — L N G P N E V K L G H L Q L A M M T S G Y L W Q N G I D E V P T S L P 9 8 t u r b o M b Q L V A - S K G M R D E V K K L P V L D F N K — L N G P N E V K L G H L Q L A M M T S G Y L W Q N G L D D V P T S L P 9 8 S u l c u l u s M b D L V A - S H K F R D A V K E M P L L D S S K — L A G Y R Q K R L A H L Q L V L I T S G Y L W Q E G E G G A V Q R L P 9 8 N o r d o t i s M b E L V A - S H K F R D A V K E M P L L D Q S K — L A G Y R Q K K L A H L Q L V L I T S G Y L W Q E G E G G A V Q R L P 9 8 m o u s e I D O V L I E - N G Q L R E E V E K L P T L S T D G — L R G H R L Q R L A H L A L G Y I T M A Y V W N R G D D D V R K V L P 1 0 8 r a t I D O K L I E - N G K L R E E V E K L P T L R T E E — L R G H R L Q R L A H L A I i G Y I T M A Y V W N R G D D D I R K V L P 1 0 8 h u m a n l D O D L I E - S G Q L R E R V E K L N M L S I D H — L T D H K S Q R L A R L V L G C I T M A Y V W G K G H G D V R K V L P 1 0 4 Y J R 0 7 8 W S L L Q E D N K V R S V I D G L D V L D L D E T I L G I > V R E L R R A Y S I L G F M A H A Y I W A S G — T P R D V L P 1 1 8 O m p h a l i u s M b N C L A A P L Y G I Y E K Y D I P P V M T Y G D 1 1 L N N S V A K G A P Q P E N I G A I I D I P G D K K 1 5 0 t u r b o M b N C L A A P L Y G I Y E K Y D I P P V M T Y G D I L L N N A I A K G G P Q P E N I S A I V D I P A D K K 1 5 0 S u l c u l u s M b E C V A K P L W N V S N D L G L K P V L T Y G D V C L T N C R V K G G D I E V M Y N L P G G - A 1 4 5 N o r d o t i s M b E C V S K P L W N V S N D L G L K P V L T F A D I C L T N C K V K N G D I E V M Y N L P G G - A 1 4 5 m o u s e l D O R N I A V P Y C E L S E K L G L P P I L S Y A D C V L A N W K K K D P N G P M T Y E N M D I L F S F P G G - D 1 6 2 r a t I D O R N L A V P Y C E L S E K L G L P P I L S Y A D C V L A N W K K K D P N G P M T Y E N M D I L F S F P G G - D 1 6 2 h u m a n l D O R N I A V P Y C Q L S K K L E L P P I L V Y A D C V L A N W K K K D P N K P L T Y E N M D V L F S F R D G - D 1 5 8 Y J R 0 7 8 W E C I A R F L L E T A H I L G V P P L A T Y S S L V L W N F K V T D E C K K T E T G C L D L E N I T T I N T F T G T - V 1 7 7 O m p h a l i u s M b E W D W F V G V S Y M S E F E F A K A V P A I Q N V F D G M D E N N D — D K I A A A L K Q I A E A V A N M Q K M M G R 2 0 8 t u r b o M b D W D W Y I G V S Y M A E F E F A K A V P A L Q N V P D G M D E N N D — D K I A A A L K Q I A E A A G N I Q K T M G R 2 0 8 S u l c u l u s M b G T E W F L K V C G L V E L T L G K G A Q S V Q N V L D G A K A N D K — A K M T S G L T E L T T T I G N M Q A A L A K 2 0 3 N o r d o t i s M b G T E K F L K V C G L V E L A F G K S G Q A I Q N V L D G A K A N D K — A K M A S G F T D L T A A I G N M Q T A L A R 2 0 3 m o u s e l D O C D K G F F L V S L L V E I A A S P A I K A I P T V S S A V E R Q D L — K A L E K A L H D I A T S L E K A K E I F K R 2 2 0 r a t I D O C D K G F F L V S L M V E I A A S P A I K A I P T V S S A V E H Q D P — K A L E K A L C S I A A S L E K A K E I F K R 2 2 0 h u m a n l D O C S K G F F L V S L L V E I A A A S A I K V I P T V F K A M Q M Q E R — D T L L K A L L E I A S C L E K A L Q V F H Q 2 1 6 Y J R 0 7 8 W D E S W F Y L V S V R F E K I G S A C I i N H G L Q I L R A I R S G D K G D A N V I D G L E G L A A T I E R L S K A L M E 2 3 7 O m p h a l i u s M b Y G E K L P I E G L F P K M W A F F G G Y G E L T L R D G L I F E G V K D Q P I K M K G G N A S Q T P T M R V L D 2 6 5 t u r b o M b F S E K L S A D V L F P K M W A F F G G Y G E L T L H D G L I F E G V K D Q P I K M K G G N A S Q S P T L R V L D 2 6 5 S u l c u l u s M b M N D N L T P D H F Y N V I i R P F L G G F G G - P A S P I S G G L I Y E G V S D A P V T M I G G S A A Q S S A M Q L L D 262 N o r d o t i s M b M N E N L T P E H F Y N G V R P F L N G F G G - P A S P I S G G L V Y E G V S D K P V T M I G G S A A Q S S T M Q V L D 262 m o u s e l D O M R D F V D P D T F F H V L R I Y L S G W K — C S S K L P E G L L Y E G V W D T P K M F S G G S A G Q S S I F Q S L D 2 7 8 r a t I D O M R D F V D P D T F F H V L R I Y L S G W K — G N P K L P E G L L Y E G V W D T P K K F S G G S A G Q S S I F Q S L D 278 h u m a n l D O I H D H V N P K A F F S V L R I Y L S G W K — G N P Q L S D G L V Y E G F W E D P K E F A G G S A G Q S S V F Q C F D 2 7 4 YJR078W M E L K C E P N V F Y F K I R P F L A G W T N M S H M G L P Q G V R Y G - A E G Q Y R I F S G G S N A Q S S L I Q T L D 2 9 6 O m p h a l i u s M b NLLGITHPQD R N A F I E E V L K Y I Q P S H R K F I Q A V A — E R Q L K G K V D A S G N A G 314 t u r b o M b NLLGIANQPE RTAFIEEIMKYIQPNHRKFIQAVG—ERMLKARVDASGNAG 314 S u l c u l u s M b NLLGVTHSPD KQAFLDEISNYMIPAHKQLLADLTKMPRKVPQIVAEAKDAN 3 1 3 N o r d o t i s M b G L L G I T H S P E KQAFLDEIRNYMPPSHKQMLADLTNMPRKVPQVVAETKDAN 313 m o u s e l D O V L L G I K H E A G K E S P AEFLQEMREYMPPAHRNFLFFLESAPPVREFVIS-RHNED 3 3 1 r a t I D O VLLGIKHDVGEGSA AEFLQEMREYMPPAHRNFLSSLESAPPVREFVIL-RRNED 3 3 1 h u m a n l D O VLLGIQQTAGGGHA AQFLQDMRRYMPPAHRNFLCSLESNPSVREFVLS-KGDAG 327 YJR078W ILIXSVKHTANAAHSSQGDSKINYLDEMKKYMPREHREFLYHLESVCNIREYVSRNASNRA 356 O m p h a l i u s M b LKEAFGALKAAVSNFRNSHVQVVTKYIVQAMEKNLPAAKQGELKPMKDS 363 t u r b o M b LKEAFQGLKAALSNLRNFHVQVVTKYIVQAFEKNLPAAKQGELKPMKES 363 S u l c u l u s M b LSKAYSGCTAALTQYRTYHIQVVTKYIVTASKSDSPKS-LAYKDTGKSD 3 6 1 N o r d o t i s M b LTKAFNGCVAAFVQYRSYHIQVVTKYIVTASKSDSPKS-LAYKDTGKSD 3 6 1 m o u s e l D O LTKAYNECVNGLVSVRKFHLAIVDTYIMKPSKKKPTDGDKSEEPSNVES 380 r a t I D O LKEAYNECVNGLVSLRMFHLSIVDTYIVKPSKQKPMGGHKSEEPSNTEN 380 h u m a n l D O L R E A Y D A C V K A L V S L R S Y H L Q I V T K Y I L I P A S Q Q P K E N K T S E D P S K L E A 376 YJR078W LQEAYGRCISMI^IFRDNHIQIVTKYIILPSNSKQHGSNKPNVLSPIEPNTKASGCLGHK 4 1 6 * * * * * * * 17 O m p h a l i u s M b t u r b o M b S u l c u l u s M b N o r d o t i a H b m o u s e l D O r a t I D O h u m a n l D O Y J R 0 7 8W RGTGGTNPMTFLRSVKDTTEKALLSWP 407 RGTGGTDVMNFLRSVKDTTKKALLSWP 407 K 6 T G 6 T D L H N F L K T V R S T T E K S L L K E 6 403 VASSKTIGTGGTRLMPFLKQCRDETVATADIKNEDKN 453 Figure 3: Sequence alignment of IDO-like myoglobins and mammalian IDOs. A m i n o a c i d s e q u e n c e s o f f o u r I D O - l i k e M b s f r o m S . diversicolor, N. madaka ( N o r d o t i s M b , D 5 8 4 1 5 ) , O . pfeifferi ( O m p h a l i u s M b , A B 0 1 7 2 5 9 ) , a n d T. cornutus ( T u r b o M b , A B 0 1 7 2 5 8 ) , a n d t h r e e m a m m a l i a n I D O s f r o m h u m a n ( X 1 7 6 6 8 ) , m o u s e ( M 6 9 1 0 9 ) , r a t ( A F 3 1 2 6 9 9 ) , a n d y e a s t p r o p o s e d I D O ( Y J R 0 7 8 W , G e n e l D : 8 5 3 5 4 1 ) w e r e a l i g n e d b y t h e C l u s t a l W 1 . 7 p r o g r a m ( C h e n n a , et al., 2 0 0 3 ) . C o n s e r v e d r e s i d u e s a r e i n d i c a t e d b y *, a n d - i n d i c a t e g a p s i n s e r t e d f o r m a x i m a l s i m i l a r i t y . 18 SidcubisMb -NordottsMb — humanlDO -mouselDO ratlDO YJR078W -OmphallusMb -turboMb Figure 4: Phylogenetic tree based on the alignment in Figure 3. Clustal W W W Service at European Bioinformatics Institute (http://www.ebi.ac.uk.clustalw) 19 Figure 5: Structure of IDO-PI complex. ( A ) O v e r a l l s t r u c t u r e o f h u m a n I D O . T h e s m a l l a n d l a r g e d o m a i n s a r e s h o w n i n b l u e a n d g r e e n r e s p e c t i v e l y . T h e c o n n e c t i n g h e l i c e s ( K - L a n d N ) a r e i n c y a n . T h e l o n g l o o p c o n n e c t i n g t h e t w o d o m a i n s i s i n r e d . T h e h e m e a n d t h e p r o x i m a l l i g a n d H 3 4 6 i s i n y e l l o w . I D O i n h i b i t o r , 4 - p h y n y l i m i d a z o l e , i s i n w h i t e . ( B ) A c t i v e s i t e o f h u m a n I D O . H e l i c e s ( f r o m A t o S ) a r e n a m e d i n t h e o r d e r o f a p p e a r a n c e i n t h e p r i m a r y s e q u e n c e . A d a p t e d f r o m S u g i m o t o et al ( 2 0 0 6 ) . 20 Figure 6: Active site of IDO-PI complex. (A) and (B) Stereo view of the residues around the heme. Two C H E S molecules bound in the distal pocket are shown in green. The 7-propionate of the heme interacts with the amino group of CHES -1 and side chain of Ser-263. From Sugimoto et al (2006). 2 1 1.5 - Physical and spectroscopic studies of IDO Ferric DDO exhibits a spectrum that is typical of heme proteins with an absorption maximum in the Soret (406 nm) and visible regions (630 nm). The ratio of absorbance at 406 to 280 nm is 2.2. Following reduction with sodium dithionite, the Soret and visible maxima shift to 420 and 560 nm. Binding of L-tryptophan to the oxygenated enzyme decreases the intensities of the absorption bands at 415, 541, 576 nm and induces a blue shift of the Soret peak in the absence of dioxygen (Yamamoto and Hayaishi, 1967). The pi of IDO as determined by isoelectric focusing is 6.9 (Littlejohn et al, 2000). The Km values for recombinant IDO are 20 uM, 5.0 uM, and 440 uM for L-tryptophan, D-tryptophan, and 5-hydroxy-L-tryptophan, respectively. The corresponding K m a x values, expressed as turnover rate, are 89, 259, and 5.3 mol product/min/mol enzyme. The O2 complex of ferrous DDO autoxidizes more rapidly in the presence of L-tryptophan. At pH 7.0 and 25°C, the autoxidation rates are 2.8 x 10"2 s"1 in the presence of tryptophan and 4.7 x 10"4 s"1 in the absence of tryptophan (Hirata, et al., 1977; Taniguchi, et al., 1979). During the catalytic cycle, DDO autoxidizes at a rate of 0.028 s"1 due to equation (3). Trp - F e 2 + - 0 2 -> F e 3 + + Trp + [0 2*] (3) Thus, in vivo the ferric enzyme must be reactivated at a corresponding rate. Prior to the current work, the mechanism responsible for this reductive reactivation was unknown. In vitro activity assays for DDO require ascorbic acid as a reducing agent and methylene blue 22 to mediate electron transfer between the enzyme and the reducing agent for optimal oxidation of L-tryptophan. Since the discovery of IDO, it has been recognized that the addition of methylene blue stimulates the formation of A^-formylkynurenine by crude enzyme preparations (Yamamoto and Hayaishi, 1967). The requirement for methylene blue was also observed with the purified enzyme, and 2 uM methylene blue is required for half-maximal activity. In addition to methylene blue, the enzyme requires ascorbic acid as a reducing agent. The concentration of ascorbic acid required for half-maximal activity is 0.2 mM, and glutathione (1 mM) or cysteine (20 mM) could not replace ascorbic acid. The enzyme is not active in the presence of hydrogen peroxide. However, IDO does not appear to be completely inactivated by exposure to peroxide because upon subsequent addition of 5 mM ascorbic acid, the enzyme was found to be ~ 60% active. Ascorbic acid could be replaced by xanthine oxidase and hypoxanthine (a superoxide generating system), especially in the presence of a small amount of catalase, and in this system methylene blue was also required. However when methylene blue (2 uM) is used in the presence of 10 mM ascorbate, no inhibition is detected even with 880 units/ml superoxide dismutase. Sono (1989) proposed that methylene blue bypasses the superoxide mediated dioxygenase activation pathway in the presence of the both ascorbate-methylene blue and xanthine oxidase-methylene blue systems. The dioxygen complex of the indoleamine 2,3-dioxygenase can be prepared at - 3 0 ° C in mixed solvents, conditions under which both autoxidation and turnover of the ternary complex are effectively suppressed (Hayaishi, et ah, 1977). Infusion of K O2 into a reaction mixture equilibrated with an atmosphere of 20% 1602, and 80% N2 23 resulted in l 8 0 incorporation into the product, but 1 6 0 was also incorporated to an extent of 30 to 50%. When KO2 was infused into the assay mixture under anaerobic conditions, the amount of the product formed was less than half the amount formed under aerobic conditions. These findings suggest that both O2" and O2 are involved in the catalytic process of this enzyme in vitro (Hayaishi, et al., 1977). The circular dichroism (CD) spectra of ferric DDO (250-700 nm) exhibited no change on addition of L-tryptophan (0.2 m M , p H 6.6) (Sono, et al., 1980). In contrast, a marked C D change was observed for the ferrous enzyme under the same treatment conditions. A marked change in the C D spectrum of the ferric enzyme was observed on addition of 5-hydroxy- L-tryptophan while the other indoleamine derivatives such as D -tryptophan, 5-hydroxy- L-tryptophan, serotonin, and tryptamine, all o f which can serve as substrates, did not induce such marked changes. The spectroscopic and spin-state changes from high-spin to low-spin induced upon binding of L-tryptophan (but not D-tryptophan) binding to ferric IDO contrasts to the case of cytochrome P450 from Pseudomonas or mammals in which absorption and E P R changes consistent with a transition from low-spin to high-spin are induced upon binding of substrate to the enzyme. Taniguchi, et al. (1979) investigated the mechanism of reaction of dioxygen and the superoxide anion radical with IDO in the ferrous and ferric states, respectively. The ferrous form of IDO was generated in situ by flash photolysis of the carbon monoxide complex of the enzyme in an air-saturated buffer. Under these conditions, IDO reacted with dioxygen to form the oxygenated enzyme that was identified on the basis of its spectrum and the isosbestic points with the ferrous enzyme. The rate constants for the binding of dioxygen, to the ferrous enzyme in the absence and presence of 0.2 m M L -24 tryptophan were determined to be 7.4 x 106 and 6.3 x 106 M" 1 s"1 at 24 ° C and pH 8.0, respectively. These authors also concluded that in the presence of the organic substrate, the ternary complex of en2yme-02-substrate can be formed either by the reaction of superoxide with the ferric enzyme or that of dioxygen with the ferrous enzyme. Once the ternary complex is formed, the reaction proceeds by use of dioxygen until the enzyme is completely oxidized to the ferric form at which point superoxide is required for formation of the ternary complex. The reaction of superoxide radical with EDO was also studied by the use of pulse radiolysis. The ferric enzyme reacted with superoxide to form the oxygenated enzyme in the absence of tryptophan. The rate constant for the reaction (8.0 x 106 M" 1 s"1 at pH 7.0) increased with decreased pH. In the presence of low concentrations of Tip (~ 50 uM), under which the catalytic site of the ferric enzyme is > 99% Trp-free at pH 7.0, the only spectroscopic species observed upon superoxide binding was Trp-bound oxygenated enzyme. So, Kobayashi, et al. (1989) concluded that under the conditions employed, superoxide binds first to the ferric enzyme to form the oxygenated enzyme followed by rapid binding of Trp (Kobayashi, et al., 1989). Papadopoulou, et al. (2005) studied the electrochemical characteristics of IDO and determined the midpoint potential for IDO to be -30 ± 4 mV at pH 7.9 and 25°C. This value is lower than that observed for myoglobins (E 0 ~ + 50 mV) but is higher than corresponding values observed for the heme peroxidases (- 100 to - 250 mV). With Trp binding, the potential increases by 46 mV, consistent with stabilization of the ferrous form of the enzyme. The treatment of recombinant IDO with peroxynitrite significantly decreases 25 enzyme activity (Fujigaki, et al., 2006). Peptide mapping analysis by liquid chromatography and electrospray ionization and tandem mass spectrometry demonstrated that residues Tyrl5, Tyr345, and Tyr353 are nitrated by peroxynitrite. The extent of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity are significantly reduced in the Tyrl5Phe variant, so it appears that nitration of Tyr 15 for IDO protein may be the most important factor in the inactivation of DDO. 1.6 - Mechanistic studies of IDO Similar to non-heme iron dioxygenases such as catechol 3,4 dioxygenase (Mendel, et al., 2004; Shu, et al., 1995), protocatechuate 4,5-dioxygenase (Arciero and Lipscomb, 1986) and quercetinase (Oka and Simpson, 1971), catalysis by IDO introduces both atoms of a dioxygen molecule across a double bond of an aromatic system and thereby breaks a carbon-carbon double bond. In contrast to monooxygenases such as cytochrome P450 and methane monooxygenase (Shteinman, 1995), which are able to oxidize alkanes, catalysis by IDO is restricted to the oxidation of carbon-carbon double bonds. Based on the results obtained from the studies with the tryptophan analogs, substituted tryptophans, and other available experimental evidence, Sono et al. (1996) reviewed two plausible reaction mechanisms for IDO: an ionic mechanism and a radical mechanism. Terentis et al. (2002) proposed that the heme-02 complex reacts by means of an ionic mechanism as shown in Figure 7. In the rate limiting step of this mechanism, an active site base (B') deprotonates the indole NH and induces nucleophilic attack by the indolic carbon 3 on the oxygen atom of the heme-02 complex (Figure 7). In this case, the 26 main role of the protein is the deprotonation of the indole NH group to "activate" the substrate. This hypothesis is supported by the fact that the basicity of the indole nitrogen significantly influences the rate of the oxygenation and because the free indole N H group is required for the IDO catalysis. After this rate limiting step, two pathways have been proposed to convert the 3-indolenylperoxy-Fe(II) intermediate into product: a Criegee-type rearrangement and a dioxetane pathway. Further rearrangements then occur to form the product N-formylkynurenine. Recently, Sugimoto et al. (2006) showed that proton abstraction by iron-bound dioxygen should be the most plausible event for the reaction mechanism of IDO because the crystal structure of the enzymes demonstrates that no potential catalytic base is present at the active site. The sole interaction of an indole N H group with the proximal oxygen atom bonded to iron should lead to electrophilic addition of a terminal oxygen atom of the bound dioxygen to the indolic carbon 3 to form the intermediate complex. A radical mechanism for IDO has also been proposed (Leeds et al, 1993) that involves a one-electron transfer between Trp (2s°' = 1,050 mV vs NHE, or 810 mV vs SCE) (DeFelippis, et al., 1989) and the heme-02 complex = - 360 mV vs SCE in dimethyl sulfoxide) (Tsang and Donald, 1990). This possibility has been discounted by the argument that such a reaction is thermodynamically unfavorable (Sono et al, 1996). 27 Figure 7: Proposed ionic reaction mechanism for conversion of tryptophan to N-formylkynurenine catalyzed by indoleamine 2,3-dioxygenase. T h e r e a c t i o n s e q u e n c e a t t h e e n z y m e a c t i v e s i t e i s i l l u s t r a t e d i n t h e f i g u r e . T w o a l t e r n a t i v e m e c h a n i s m s f o r i t s c o n v e r s i o n t o t h e p r o d u c t a r e s h o w n : ( a ) C r i e g e e -t y p e r e a r r a n g e m e n t a n d ( b ) d i o x e t a n e p a t h w a y s a f t e r t h e f o r m a t i o n o f t h e 3 -i n d o l e n y l p e r o x y - f e r r o u s i n t e r m e d i a t e . A d a p t e d f r o m S o n o e f a l , 1 9 9 6 . 28 1.7-IDO inhibitors Muller, et al. (2005b) have recently presented a comprehensive review of IDO inhibitors that emphasizes the primary focus of previous work on derivatives of indole and fi-B-carboline, a heterocycle that is structurally related to tryptophan (see Table 5). Inhibitors that exhibit half-maximal inhibition at sub-micromolar concentration either in vivo or in vitro have not been reported (Muller, et al., 2005b). The inhibitors 1-methyl-DL-Trp, (5-(3-benzofuranyl)- DL-alanine (the oxygen analog of Trp), and p-[3-benzo(b)thienyl]- DL-alanine (the sulfur analog of Trp) were found to be competitive (K\ ~ 7-70 uM) with respect to Trp for rabbit small intestinal IDO (Cady and Sono (1991)). These authors also studied the spectroscopic changes that occur upon binding of these inhibitors to the native ferric, ferrous, ferrous-CO, and ferrous-NO enzyme. The changes observed upon binding of 1-methyl-DL-Trp to IDO are similar to those observed upon binding of the substrate; while the sulfur and the oxygen analogs of Trp exhibit relatively small effects except for the influence of P-[3-benzo(b)thienyl]- DL-alanine on the CD spectrum. Each of these three Trp analogs competes with the substrate for the ferrous-CO enzyme, a model for the ferrous-02 enzyme. Recently, Gaspari et al (2006) identified the natural product brassinin (Table 5) as a moderate inhibitor of IDO using a screen of indole-based structures. These authors undertook a structure-activity study to determine which elements of the brassinin structure could be modified to enhance potency. Three important discoveries resulted: (i) The dithiocarbamate portion of brassinin is a crucial moiety that binds to the heme iron. (ii) An indole ring is not necessary for IDO inhibition. 29 Table 5: Representative IDO inhibitors. I n d o l e o r p y r r o l l i k e r i n g s a r e i n d i c a t e d i n r e d . N A M E S T R U C T U R E K\ R E F E R E N C E 1-methyl-tryptophan M T H -tryptophan brassinin P-carboline 4-phenyl imidazole brassilexin 34 uM (Cady and Sono, 1991) 11.4 uM (Muller, et al., 2005a) 97.7 uM (Gaspari, et al, 2006) 178 uM (Eguchi, etal., 1984) 4.4 uM (Sono and Cady, 1989) 5.4 uM (Eguchi, et al., 1984) 30 (iii) Substitution of the S-methyl group of brassinin with large aromatic groups provides inhibitors that are three times more potent than 1 -methyl-tryptophan in vitro. The effects of norharman, a P-carboline, and of 4-phenylimidazole on the catalytic and spectroscopic properties (optical absorption, CD, and magnetic CD) of the rabbit small intestinal IDO were investigated by Sono and Cady (1989) in assays using L -or D-tryptophan as substrates and the ascorbic acid-methylene blue cofactor system (25 °C). Both norharman and 4-phenylimidazole exhibited noncompetitive inhibition with respect to L-tryptophan and D-tryptophan. Norharman exhibited similar dissociation constants (Ku = ~ 10 uM at pH 7.0) with ferrous and ferric EDO. Norharman binding induces low-spin complexes for the ferric and ferrous IDO. Very similar spectroscopic changes were observed with the binding of 4-phenylimidazole and norharman to the reduced EDO. From these experiments, Sono and Cady (1989) concluded that norharman interacts directly with heme iron as a nitrogen donor ligand, competing with oxygen for the heme iron of the reduced (active) enzyme. Instead, 4-phenylimidazole appears to compete for the heme-binding site in the ferric enzyme and displays slight negative cooperativity on binding to the ferrous enzyme (Sono and Cady, 1989). It has been noted that IDO and TDO are not inhibited similarly by the same compounds (Eguchi et al, 1984). For example, norharman is uncompetitive (K\ = 0.12 mM) with respect to L-tryptophan for rabbit intestinal IDO, while it is linearly competitive (Kx - 0.29 mM) with L-tryptophan for mouse liver TDO. Some (J-carbolines selectively inhibit one or the other of these dioxygenases but not both. In addition, the pseudomonad TDO is inhibited by a different spectrum of P-carbolines. This enzyme specificity becomes more evident when indolic compounds are considered. For example, 31 indole-3-acetamide, indole-3-acetonitrile and indole-3-acrylic acid exhibit potent inhibition of mammalian TDO, but they are weak inhibitors of the pseudomonad TDO, and they exhibit no inhibition of IDO. Eguchi et al (1984) concluded that these results imply different active site structures for enzymes from various sources. Notably, the compound 680C91 ((E)-6-fluoro-3-[2-(3-pyridyl)vinyl]-lH-indole) (Figure 8) was reported to be a potent (AT, = 51 nM) and selective competitive inhibitor of rat liver TDO inhibitor (with respect to Trp) that does not inhibit IDO, monoamine oxidase A and B, 5-hydroxytryptamine uptake or 5-hydroxytryptamine 1A, ID, 2A and 2C receptors at a concentration of 10 uM (Salter, et al., 1995). In general, however, most recently discovered inhibitors of IDO activity have not been characterized in terms of their ability to inhibit TDO from mammalian sources largely as the result of difficulty in obtaining sufficiently purified samples of TDO. At present, recombinant expression of mammalian TDO appears to be compromised by the greater difficulty inherent in efficient bacterial expression of a heterologous tetrameric enzyme, but perhaps this limitation will be overcome with time. The IDO inhibitors 1-methyltryptophan and methyl-thiohydantoin-tryptophan have been used to demonstrate proof of principle of the value of IDO inhibition in cancer therapy although their selectivity towards EDO has not been thoroughly investigated in vivo. Administration of these compounds concurrently with chemotherapeutic agents that are by themselves therapeutically ineffective resulted in slowing the growth of tumors in mice (Muller, et al, 2005a; Uyttenhove, et al, 2003). Notably, 1-methyltryptophan is more active against EDO in vitro (K\ - 35 uM) than in a cell-based EDO assay (ECso= 200 uM) (Muller, et al., 2005a). 1-methyltryptophan is also poorly soluble and difficult to 32 Figure 8: Inhibitor 680C91 ((E)-6-fluoro-3-[2-(3- pyridyl)vinyl]-1H-indole) of tryptophan 2,3-dioxygenase (TDO). 33 administer to animals (Muller, et al., 2005b; Uyttenhove, et al, 2003). Methyl-thiohydantoin-tryptophan is more potent than 1-methyltryptophan in vitro (K\ = 11 u M ) and in a cell-based assay (EC5o= 12.5 u M ) (Muller, et al., 2005a). This inhibitor is also more soluble but more rapidly cleared from plasma (Muller, et al., 2005a). These results were attributed to the ability of even poor IDO inhibitors to impede the T-cell immune response sufficiently that otherwise poor chemotherapeutic agents are potentiated. A s for any other heme containing protein, EDO is also inhibited by small, anionic inhibitors that bind directly to the ferriheme (e.g., cyanide, azide, fluoride) and by diatomic gaseous ligands that bind directly to ferroheme (carbon monoxide and nitric oxide). These inhibitors bind with high affinity, but they exhibit essentially no selectivity for specific heme proteins or enzymes. A s a result, these compounds have well known toxicities. Nevertheless, these inhibitors are useful for mechanistic investigations (e.g., Sono (1989)). 1.8 - Uncatalyzed oxidation of tryptophan Tryptophan can be converted to JV-formyl kynurenine without EDO (Figure 9); however, the conditions required for this transformation cannot be achieved in cytoplasm. The oxygenation of tryptophan can be carried out by irradiation (hv > 550 nm) at 0.5 °C in the presence o f methylene blue overnight. Tryptophan reacts with singlet oxygen produced by the dye-sensitized photooxidation in aqueous solution to give the tricyclic hydroperoxide 4 with 85% yield. Similar oxygenation of tryptophan in a buffer solution of N a 2 C 0 3 - A c O H (pH 7) affords N-formylkynurenine 3 with a 55% yield. 34 OOH X NH NH ^ C 0 2 H H 2 0 ^ OOH ^ ^ ^ C 0 2 H hv/sens/02 , C 0 2 H NH2 L Tryptophan photosensitizer: Methylene blue Rose Bengal \ Buffer solution N a 2 C0 3 AcOH pH 7 N a 2 C0 3 AcOH pH 7 3 Figure 9: Uncatalyzed oxidation of tryptophan. A d a p t e d f r o m N a k a g a w a et al., 1 9 7 7 . 3 5 A large variety of other secondary products are obtained in the reaction mixture as the result of oxidation of tryptophan at positions other than 3. Clearly, this uncatalyzed reaction has very low specificity and selectivity. 1.9 - Goals of the current study Muller et al (2005b) concluded that "one important goal in the development of IDO inhibition as a cancer therapy will be to discover more potent inhibitors, and it seems that diversification of IDO inhibitor structures may be necessary to achieve this goal." Accordingly, the primary goal presented in this dissertation is to identify and characterize new inhibitors of human EDO. Some of this work has been published prior to the completion of this dissertation. Most of the known IDO inhibitors are tryptophan analogues, which are active only at concentrations of -10 | i M and greater, making them marginal drug candidates. As part of a program designed to identify more potent IDO inhibitors belonging to new structural classes, a library of marine invertebrate extracts was screened for the ability to inhibit IDO in vitro and in yeast. These inhibitors were isolated by activity-guided fractionation from these extracts, and they were then studied individually to determine the basis for their inhibition of TDO and their potentials in drug therapy in collaboration with Alban Pereira in the group of Professor Raymond Andersen (Pereira et al, 2006; U.S. patent application (2004) PCT/CA2005/001087). To provide an initial means by which the new TDO inhibitors discovered in this work could be evaluated in vivo, an assay based on the inhibition of yeast growth was implemented in collaboration with Aruna Balgi in the group of Professor Michel 36 Roberge. Expression of a human gene in yeast can often produce a quantifiable phenotype, and screening for chemicals that reverse the phenotype has led to the identification of selective inhibitors of the protein expressed. Expression of IDO by cells transformed with the IDO gene was confirmed by immunoblots with rabbit anti-IDO antibodies that were the result of this research work (Vottero et al., 2006a). While the development of a high-throughput activity screen for IDO activity in vivo was essential to identification of these new inhibitors, the concomitant development of a high-throughput activity screen for TDO activity in vivo permitted identification of cytochrome bs as a possible electron transfer partner for IDO in vivo (Vottero et al., 2006b). The availability of high throughput assays for IDO activity both in vivo and in vitro permits the design of a variety of informative experiments that identify a number of new directions for study in which physical (kinetic, thermodynamic and spectroscopic) methods can be employed to understand the structural and mechanistic basis for the catalytic and physiological activities of this enzyme. 37 CHAPTER II - MATERIALS AND METHODS 2.1 - Reagents. Methyl-thiohydantoin-tryptophan (MTH-tryp) and other synthetic EDO inhibitors were purchased from Sigma and solubilized in D M S O . 1-Methyl-tryptophan was solubilized in 100 m M HC1 in water. The N C I Diversity Set library and all compounds described by their N S C designation were obtained from the National Cancer Institute (Holbeck, 2004). A l l other reagents were products of Sigma. 2.2 - Biological materials. The library of 4800 extracts prepared from marine invertebrates that was screened for the presence of IDO inhibitors in this work is maintained by Prof. Raymond Andersen and his research group in the Departments of Earth and Ocean Science and Chemistry at this University. Samples of the hydroid Garveia annulata were collected by hand using S C U B A in Barkley Sound, British Columbia, at a depth of 10-20 m by members of Prof. Andersen's research group. 2.3 - Expression and purification of recombinant human indoleamine 2,3-dioxygenase. The c D N A corresponding to the complete open reading frame of IDO was amplified from a permanent human cell line (Edgell et al, 1983) by Michael Page in Professor MacGil l ivray 's laboratory using P C R and was verified by sequence analysis. The coding region for human IDO (Figure 11) was cloned into the pET-28a vector (Novagen) between the EcoRI and X h o l restriction site (Figure 10). Recombinant enzyme was expressed in transformed E. coli BL21 (star) cells, grown in T B media containing kanamycin (50 mg/L), chloramphenicol (34 mg/L) and 75 u L / L 10% antifoam 3 8 solution. D?TG was added when the OD600 reached a value of 1. After overnight growth, the cells were separated by centrifugation at ~ 2,700 g, washed with 50 m M K P B p H 7 and stored at -80°C. Frozen cells were resuspended in 150 m L of 50 m M K P B p H 8, 300 m M N a C l and 20 m M imidazole. Cells were broken using a French Press. The lysate was then centrifuged at ~ 11,000 g (1 hour, 4°C). The supernatant fluid was applied to a N i - N T A column (10 cm x 2.5 cm). The column was washed with 400 m L of 50 m M K P B p H 8, N a C l (300 m M ) and imidazole (20 m M ) . Recombinant protein was then eluted with 100 m M imidazole. Thrombin (0.1 mg/mg of protein, Novagen) was added to the samples with A406 / A280 > L Samples were dialyzed against 20 m M T r i s H C l p H 7.5 and then applied to a Mono-Q column (20 cm x 2.5 cm) that had been equilibrated with 16 m M T r i s H C L pH7.0 and I m M D T T . A p o IDO eluted first with no salt added and holo IDO eluted immediately after a salt gradient was applied (from 0 m M to 300 m M N a C l in 16 m M T r i s H C L pH7.0 in a total volume of 100 mL) . The purified native ferric enzyme exhibited an A405/A280 value of 2.1 at p H 6.0 (20mM K P B , 25 °C). Enzyme concentration is expressed in terms of heme content on the basis of absorbance at 405 nm (e = 159 mM" 1 cm"1 at p H 6.0 and 25 °C (Sono & Dawson, 1984)). The yield of IDO protein was approximately 25 mg/L of T B growth media. 39 FcoRI-5 Spkl 6159 PflfiU 6051 &JAP1.5950 7bgII-S724 MM-5626 Ajna 5626 Se/I-5612 TogD - 5506 fioED. 5444 .4j>al 5423 AyOMI-5419 fisrHH 5215 FcoRV 5180 «pal- 5124 HincH- 5124 fisnXI 4988 fisoXI 4958 i»s*AI - 4785 Bgfl 4569 Fspl 4548 ' •FspAI-4548" PpiiMI - 4520 " SJIKIOI-4420" fispEl 4336 Mel 6518 Ad*l 6513 Ncol 6453 — 6414 Bgin 6348 SgrAI-630r, Bam HI 6551 6522 ftil-pET 28a+IDO 6553 bp A'IKKI 3971 TthA 111 - 3783 •KraAl - 377 BWZ17I-3758 /led-37 Sapl 3642 PciI-3525 .4ffin-3525 SsSI-3352 .«wNI-3116 117 Mfel 168 fifpI-203 Scfl-211 .4JMI 229 — Bm^BI 248 Wcol 270 AH«I-277 R i l l II-395 Bom HI 421 -4ioI 446 Alol - 478 Pstl 488 ' ftrl 746 ' ArI-778 ' ZraI-832 4offl-834 ' Hintm 999 I £m36I 1034 "SSBRI-1051 HIKCII- 1220 'flpaI-1220 'Aral 1223 '.Y*oI-I223 ' Blpl 1301 'SspQ 1378 1 Bsa.M 1626 £>rom- 1629 ' Alol 1665 firaXI 1665 ' Pptl-1665 SsoXl 1695 .4JoI-1697 ' PflMl 2067 Bj.ul 01 2307 4HSI-2329 Pnd 2329 'fiswl 2368 ' &wl - 2445 4voI 2451 Jbtf-2451 Anal 2453 Ctol 2634 Amrf 2670 fpil-2819 ^11-2851 Figure 10: Functional map of the 6.553 kbp plasmid pET 28 - IDO. S e l e c t e d r e s t r i c t i o n s i t e s a n d g e n e s e n c o d e d i n t h e p l a s m i d a r e i n d i c a t e d i n t h e f i g u r e . 4 0 1 ATGGCACACG CTATGGAAAA CTCCTGGACA ATCAGTAAAG AGTACCATAT M A H A M E N S W T I S K E Y H I 51 TGATGAAGAA GTGGGCTTTG CTCTGCCAAA TCCACAGGAA AATCTACCT6 D E E V G F A L P N P Q E N L P D Ps i l BsrDI 101 ATTTTTATAA TGACTGGATG TTCATTGCTA AACATCTGCC TGATCTCATA F Y N D W M F I A K H L P D L I Plel MscI Mlyl Eael TaqI Nspl BlpI 151 GAGTCTGGCC AGCTTCGAGA AAGAGTTGAG AAGTTAAACA TGCTCAGCAT E S G Q L R E R V E K L N M L S I HinPlI Hhal Haell Tsp45I Be l l Ahdl Maelll BmgBI Bfal 201 TGATCATCTC ACAGACCACA AGTCACAGCG CCTTGCACGT CTAGTTCTGG D H L T D H K S Q R L A R L V L G Styl Ncol Nsil Btgl BfrBI BsaJI Ndel TspGWI 251 GATGCATCAC CATGGCATAT GTGTGGGGCA AAGGTCATGG AGATGTCCGT C I T M A Y V W G K G H G D V R Sspl BstXI 301 AAGGTCTTGC CAAGAAATAT TGCTGTTCCT TACTGCCAAC TCTCCAAGAA K V L P R N I A V P Y C Q L S K K T t h l l l l 351 ACTGGAACTG CCTCCTATTT TGGTTTATGC AGACTGTGTC TTGGCAAACT L E L P P I L V Y A D C V L A N W Alwl NlalV BstYI BamHI Alol Alwl Alol 401 GGAAGAAAAA GGATCCTAAT AAGCCCCTGA CTTATGAGAA CATGGACGTT K K K D P N K P L T Y E N M D V Sfcl Bed PstI T f i l BsaX 451 TTGTTCTCAT TTCGTGATGG AGACTGCAGT AAAGGATTCT TCCTGGTCTC L F S F R D G D C S K G F F L V S PvuII MspAlZ Tsp509I 501 TCTATTGGTG GAAATAGCAG CTGCTTCTGC AATCAAAGTA ATTCCTACTG L L V E X A A A S A I K V I P T V 4 1 BsmFZ HinPlI BslFZ Hhal BsrDI BslFZ HaeXZ Mmel 551 TATTCAAGGC AATGCAAATG CAAGAACGGG ACACTTTGCT AAAGGCGCTG F K A M Q M Q E R D T L L K A L 601 TTGGAAATAG CTTCTTGCTT GGAGAAAGCC CTTCAAGTGT TTCACCAAAT L E 1 A S C L E K A L Q V F H Q I Hpy8Z 651 CCACGATCAT GTGAACCCAA AAGCATTTTT CAGTGTTCTT CGCATATATT H D H V N P K A F F S V L R I Y L PsrZ BseYI Hpyl88I PsrZ 701 TGTCTGGCTG GAAAGGCAAC CCCCAGCTAT CAGACGGTCT GGTGTATGAA S G W K G N P Q L S D G L V Y E BsgX BtsZ 751 GGGTTCTGGG AAGACCCAAA GGAGTTTGCA GGGGGCAGTG CAGGCCAAAG G F W E D P K E F A G G S A G Q S ZraZ BsaHX Hgal A a t l l BseYI Xcml 801 CAGCGTCTTT CAGTGCTTTG ACGTCCTGCT GGGCATCCAG CAGACTGCTG S V F Q C F D V L L G I Q Q T A G Pfol Nspl Bpml 851 GTGGAGGACA TGCTGCTCAG TTCCTCCAGG ACATGAGAAG ATATATGCCA G G H A A Q F L Q D M R R Y M P Bspl286I Plel BsiHKAI Mlyl TspGWI 901 CCAGCTCACA GGAACTTCCT GTGCTCATTA GAGTCAAATC CCTCAGTCCG P A H R N F L C S L E S N P S V R FauZ HgaZ AciZ HindZZZ BsaHZ 951 TGAGTTTGTC CTTTCAAAAG GTGATGCTGG CCTGCGGGAA GCTTATGACG E F V L S K G D A G L R E A Y D A BseRI Tsp45Z Bsal Bsu36I Bed Maelll 1001 CCTGTGTGAA AGCTCTGGTC TCCCTGAGGA GCTACCATCT GCAAATCGTG C V K A L V S L R S Y H L Q Z V TatZ TfiZ 1051 ACTAAGTACA TCCTGATTCC TGCAAGCCAG CAGCCAAAGG AGAATAAGAC T K Y I L Z P A S Q Q P K E N K T AcuZ Hpyl88Z Bpml 1101 CTCTGAAGAC CCTTCAAAAC TGGAAGCCAA AGGAACTGGA GGCACTGATT S E D P S K L E A K G T G G T D L 4 2 Tsp509I EcoNI A p o l AcuX T a t I B s l I 1151 TAATGAATTT CCTGAAGACT GTAAGAAGTA CAACTGAGAA ATCCCTTTTG M N F I i K T V R S T T E K S L L 1201 AAGGAAGGTT AA K E G * Figure 11: Sequence of the IDO cDNA. S e v e r a l r e s t r i c t i o n s i t e s a r e i n d i c a t e d . 4 3 2.4 - In vitro IDO activity assay (discontinuous method): IDO activity was determined as described elsewhere (Takikawa, et al., 1988) with some modifications. The reaction mixture (1 mL) contained KPB (50 mM, pH 6.5), ascorbic acid (20 mM), catalase (200 mg/mL), methylene blue (10 mM), L-tryptophan and recombinant EDO. The reaction was carried out at 37 °C for 30 min and stopped by the addition of trichloroacetic acid (200 u.1 of a 30 % (w/v) solution). After heating at 65 °C for 15 min, the reaction mixtures were centrifuged at maximum speed in a microcentrifuge for 10 min, and the supernatant fluid was mixed with />-dimethylaminobenzaldehyde in acetic acid (1.2 ml of 2 % (w/v) solution) to convert any kynurenine present to kynurenine, N-p-dimethylaminobenzylidene. The amount of this product was monitored at 480 nm as a measure of EDO activity. The reactions involved in this assay are shown in Figure 12. 2.5 - In vitro IDO activity assays (continuous method) for kinetic parameter determination: Kinetic parameters for EDO were determined at 25°C with the assay described by (Takikawa, et al, 1988) with minor modifications. The reaction mixture consisted of KPB (100 mM, pH 6.5), methylene blue (25 pM), bovine liver catalase (200 pg), ascorbic acid (10 mM), EDO (50 nM), and various concentrations of tryptophan and inhibitors. The rate of N-formylkynurenine formation (A£32inm - 3.75 mM"1 cm"1) was determined from the slope of the initial linear absorbance increase at 321 nm as a function of time. 2.6 - In vitro IDO activity assay (end-point method) for screening natural product and chemical libraries: Microtitre plates (96 well) were filled with the 44 reaction mixture (80 uL) containing tryptophan (1 m M ) and no enzyme using an R D X microtiter dispensing system (Dynex Technologies). Solutions o f library samples were transferred from stock plates to assay plates containing yeast with a BioRobotics T A S 1 robot fitted with a 96-pin (0.4-mm diameter each) tool, each pin of which transfers about 20 n L of solution. The reaction mixture (20 | i L ) containing the enzyme was then added to each well with an R D X Microtiter Dispensing System (Dynex Technologies). The Microtitre plates prepared in this manner are shown in Figure 13. The reaction was stopped by the addition of trichloroacetic acid (20 uL , 30 % (w/v) solution) to each well . After heating the plates at 65 °C for 15 minutes,/»-dimethylaminobenzaldehyde in acetic acid (120 U.L of 2% (w/v) solution) was added to each well . The amount of yellow product was monitored at 480 nm as a measure of IDO activity with a Tecan Sunrise plate reader. In total, ~ 4500 extracts from the marine invertebrate library were screened. The reactions relevant to this assay are shown in a following Figure 12. 2.7 - Yeast strains and vectors: Human IDO was expressed in yeast strain EIS20-2B (MATa, ade2-l, his3-ll, 15 leu2-3, 112 trpl-1, ura3-l, canl-100, pdr5A, snq2A), lacking the major efflux pumps PDR5 and SNQ2; the expression vector p A R C 2 5 B has been described by Burke, et al., (2000). The c D N A corresponding to the complete open reading frame of human IDO was amplified by P C R with the following primers: 5' C A A A A A A T T G T T A A T A T A C C T C T A T A C T T T A A C G T C A A G G G A A T A T A a t g g c a c a c gctatggaaaactcctgg-3' and 5 ' C T T G C G G G G T T T T T C A G T A T C T A C G A T T C A T A G A T C T C T C G A G T T A a c c t t c c t t c a aaaggatttctcagttg-3', 45 o 2 coo-NH-COH C H 3 C H 3 K«r 480 nm H C H 3 C H 3 Dimethylamino benzaldehyde TCA + 60 °Cfor 10 mim NH 2 Figure 12: Chemical reactions related to the in vitro assay described in Section 2.7 4 6 Control • Figure 13: Screen plate with inhibitors from the natural library. C o n t r o l c o l u m n i s i n d i c a t e d i n f i g u r e : + w e l l s c o n t a i n I D O , - w e l l s d o n o t c o n t a i n e n z y m e . A n e x t r a c t w i t h i n h i b i t o r y c a p a b i l i t y i s i n d i c a t e d w i t h a b l a c k s q u a r e . 47 where capital letters hybridize to the vector and lower case letters hybridize to the 5' and 3 ' ends of the IDO coding sequence. P C R amplification was carried out with the VentR® D N A polymerase (New England BioLabs Inc.) according to the manufacturer's instructions. The P C R products and p A R C 2 5 B linearized with BamHI and Xhol were transformed into yeast with the lithium acetate. Transformed cells were plated in minimum synthetic medium with 2% glucose and without leucine and were grown at 30 °C. Colonies were then analyzed for the correct insertion of the EDO gene in p A R C 2 5 B by colony P C R . Plasmids were rescued from selected colonies into E. coli (Tugendreich et al, 2001), digested with Sfil, and the resulting IDO expression cassette was introduced as a single copy into the LYS2 locus of EIS20-2B. D N A sequences of all constructs were determined to verify the absence of random mutations. Cloning of the His346Gly IDO mutant was carried out as above except that the c D N A corresponding to the complete open reading frame of IDO was amplified by P C R with the following primers: 5'-cttatgacgcctgtgtgaaagctctggtctccctgag-gagctacggtctgcaaatcg-3' and 5'-C T T G C G G G G T T T T T C A G T A T C T A C G A T T C A T A GATCTCTCGAGTTAaccttccttcaaaagggatttctcagttg-3' where the underlined letters correspond to the His346Gly mutation. For chromosome integration, I D O - p A R C 2 5 B was linearized with Bsu36i and Xhol. 2.8 - GFP-IDO construct: To clone GFP-tagged IDO, the c D N A corresponding to the complete open reading frame of G F P was amplified by P C R from pRSETb G F P S65T with the following primers: 5'-A G A C T G T A A G A G T A C A A C T G A G A A A T C C C T T T T G A A G G A A G G T G G T a t g g c t a g c a 48 tgactggtggacagcaaatggg-3where the capital letters pair to the end of the IDO coding sequence, the lower case letters pair to the beginning of the G F P coding sequence, and the underlined letters encode two glycines that act as a connector between these two proteins; and 5 - C T T G C G G G G T T T T T C A G T A T C T A C G A T T C A T A G A T C T C T C G AGTTatttgtagagccatccatgccatgtg- 3 ' where the capital letters pair to p A R C 2 5 B and the lower case letters pair to the end of the G F P coding sequence. 2.9 - In vivo IDO activity assay based on yeast growth inhibition: Expression of a human gene in yeast can often produce a quantifiable phenotype, and screening for chemicals that reverse the phenotype has led to the identification of selective inhibitors of the protein expressed (Kurtz, et al., 1995; Perkins, et al., 2001; Simon and Bedalov, 2004). To provide an initial means by which the new IDO inhibitors discovered in this work could be evaluated in vivo, a yeast growth strategy of this type was implemented with the technical assistance of Aruna Balgi in Professor Michel Roberge's laboratory. Yeast containing the integrated human EDO expression cassette (mutant or wi ld type) or empty vector (control) were grown overnight in complete synthetic medium with 2% glucose and without leucine (repressed condition). The following day, cells were washed twice with synthetic medium lacking leucine, tryptophan and sugar, and they were then diluted to a final A6oonm o f 0.02 in synthetic medium containing galactose (2%) and tryptophan (25 u M ) but lacking leucine unless otherwise indicated (induced condition). Diluted cells (100 uL) were added to each well o f 96-well clear polystyrene plates. After 40-45 h at 30 °C, plates were shaken to resuspend the cells, and A6oonm was measured with a Tecan Sunrise plate reader. A l l assays were carried out in triplicate, and data 49 shown are averages of three experiments with the corresponding standard deviations. Compounds from the NCI Diversity Set were transferred from stock plates (lOmM in DMSO) to plates containing yeast with a BioRobotics TAS1 robot fitted with a 96-pin (0.4- mm diameter) tool that transfers -20 nL of solution. Samples that promoted yeast growth (that inhibited IDO) were verified in a dose response curve using the strain containing integrated IDO and the strain containing the integrated empty vector as a control. DMSO concentration was kept constant at 0.1% in all wells for all experiments. 2.10 - Libraries of random IDO variants: Randomly-mutated forms of recombinant IDO were produced by error-prone PCR by the method of Miyazaki and Takenouchi (2002). In short, PCR amplification of the TDO gene was performed in Tris-HCl buffer (10 mM, pH 8.3) containing 50mM KC1 (50 mM), MgCl 2 (7 mM), dATP (0.2 mM), dGTP (0.2 mM), dCTP (1 mM), dTTP (1 mM), primers (25 pmol), plasmid (50 nM), and Taq DNA polymerase (2.5 units)(Stratagene) in a total volume of 50 pL. The cDNA corresponding to the complete open reading frame of human IDO was amplified by PCR with the following primers: 5' - C A A A A A A T T G T T A A T A T A C C T C T A T A C T T T A A -CGTCAAGGGAATATAatggcacacgctatggaaaactcctgg-3' and 5 C T T G C G G G G T T -TTTCAGTATCTACGATTCATAGATCTCTCGAGTTAaccttccttcaaaaggatttctcagttg-3' where capital letters hybridize to the vector and lower case letters hybridize to the 5' and 3' ends of the IDO coding sequence. The PCR products and pARC25B linearized with BamHI and Xhol were transformed into yeast by the lithium acetate procedure. Transformed cells were plated in minimum synthetic medium containing glucose (2%) but lacking leucine and were grown at 30 °C. Screening the resulting randomly generated 50 EDO variants for activity was performed as indicated in section 2.9. 2.11 - Cytochrome bs and IDO interaction in yeast: The yeast expression vector p A R C 2 5 B , which contains a Gal4 promoter and a L e u + marker, has been described elsewhere (Burke, et al., 2000). A l l reagents were obtained from Aldr ich except for bovine liver catalase (Sigma, C-100). The yeast deletion set (BY4741 MATSL ura3A0 leu2A0 h is3Al met 15AO) was obtained from Open Biosystems, and gene disruptions were verified by analysis of genomic D N A by P C R . Yeast tryptophan auxotrophs were produced by URA3 based conversion of the TRP1 locus using plasmid p N K Y 1 0 0 9 (Alani , et al, 1987). The parental yeast strain has a BY4741 M 4 7 a ura3A0 leu2A0 h is3Al met l5A0 trplAura3 genotype. The c D N A corresponding to the complete open reading frame of EDO was amplified by P C R with the following oligodeoxyribonucleotides: 5'-C A A A A A A T T G T T A A T A T A C C T C T A T A C T T T A A C G T C A A G G G A A T A T A a t g g c a c a c gctatggaaaactcctgg-3' and 5' - C T T G C G G G G T T T T T C A G T A T C T A C G A T T C A T A -GATCTCTCGAGTTAaccttccttcaaaagggatttctcagttg-3' where the capitalized bases pair to the p A R C 2 5 B vector and the bases in lower case hybridize to the beginning and end of the IDO O R F . P C R amplification was carried out with VentR® D N A polymerase (New England BioLabs Inc.) according to the manufacturer's instructions. The P C R products were transformed into yeast by the lithium acetate procedure, and p A R C 2 5 B was linearized by BamHI and X H O . Transformed cells were plated in minimum medium with 2% glucose without leucine and kept at 30 °C. Colonies were then checked for the correct construct by colony P C R . Plasmids were rescued from 51 selected colonies into E. coli by standard procedures. All constructs were sequenced to verify the absence of random mutations. 2.12 - Construction of the His346Gly IDO variant: The cDNA corresponding to the complete open reading frame of DDO was amplified by PCR with the following oligodeoxyribonucleotides: 5'-cttatgacgcctgtgtgaaagctctggtctccctgaggagctacggtctgcaaatcg-3' and 5'-CTTGCGGGGTTTTTCAGTATCTACGATTCATAGATCTCTCGAGTTAaccttcttcaaaa gggatttctcagttg-3' where the capitalized bases hybridize to the pARC25B vector, the bases in lower case hybridize to the IDO ORF, and the underlined bases introduce the His346Gly mutation. PCR amplification was carried out as indicated above. Yeast were transformed with the PCR products and IDO-pARC25B linearized by Bsu36i and Xhol enzymes by the lithium acetate procedure. Other methods were as indicated above. All constructs were sequenced to verify the presence of the desired mutation and absence of random mutations. Yeast strains containing integrated IDO (mutants or wild type) and empty vector (control) were grown overnight in synthetic medium containing glucose (2%) and lacking leucine. The following day, cells were washed twice with synthetic medium lacking leucine, tryptophan and sugars and then diluted to a final ODgoonm of 0.02 in synthetic medium with galactose (2%) but lacking leucine and tryptophan. The diluted cells (100 (iL) were added to 96-well plates containing varying amounts of tryptophan. After 40 to 45 hours at 30 °C, these plates were shaken to resuspend the cells inside the wells, 52 and ODeoonm values were measured with a Tecan Sunrise plate reader. Growth curves were determined in triplicate, and curves shown are averages of three experiments. 2.13 - Preparation of yeast cell lysate: Yeast cultures (25 mL, 40 h) were grown on synthetic medium containing galactose (2%), tryptophan (2 mM) and lacking leucine. Cells were harvested by centrifugation, and cell pellets were lysed with glass beads. Acid-washed glass beads (425-600 um, Sigma, St. Louis, MO) and lysis buffer (50 mM KPB, pH 6.5; 100 mM NaCl; 1 mM EDTA, IX protease inhibitor cocktail) were added to the cell pellets, and the mixture was vortexed three times for 7 min each, with 3 min incubations on ice in between. Glass beads and cell debris were removed by centrifugation, and protein extracts were quantified with the Bradford assay (BioRad, Hercules, CA). DDO activity of the cell lysates was determined as described elsewhere (Takikawa, et al., 1988) with some modifications. The reaction mixture (1 mL) contained potassium phosphate buffer (50 mM, pH 6.5), ascorbic acid (20 mM), catalase (200 units / ml), methylene blue (10 mM), L-tryptophan (1 mM), and cell lysate proteins (1 mg in ~50 ul). The reaction was carried out at 37 °C for 30 min and stopped by the addition of trichloroacetic acid (200 uL of a 30 % (w/v) solution). After heating at 65 °C for 15 min, the reaction mixtures were centrifuged at maximum speed in a microcentrifuge for 10 min, and the supernatant fluid was mixed with p-dimethylaminobenzaldehyde in acetic acid (1.2 mL of 2% (w/v) solution). The amount of this product was monitored at 480 nm as a measure of EDO activity. 53 2.14 - Western blots: After obtaining protocol approval from the U B C Animal Care Committee, polyclonal anti-DDO antiserum was obtained by immunizing New Zealand White rabbits with purified recombinant human IDO (lacking the His6-tag) at the U B C Animal Care Facility. Members of the Animal Care Facility staff performed all procedures related to animal handling. Western blots were prepared by standard procedures involving 10 % S D S - P A G E of cell lysate (100 u.g total protein). In brief, a semi-dry transfer (Biorad; Transblot SD) was used to transfer the protein samples to nitrocellulose membranes according to the manufacturers' recommendation. The membrane was washed in d t ^ O prior to being blocked with a solution of 10% non-fat mi lk in T B S containing 0.1% Tween-20 (5%-TBS-T) (overnight at 4 °C). The membrane was incubated with rabbit polyclonal anti-IDO antiserum (1:75,000 dilutions in 5%-TBS-T, 2 hours at room temperature). The membrane was then washed extensively with 5%-TBS-T prior to being incubated with the appropriate secondary antibody (sheep anti rabbit l g horseradish peroxidase conjugated antibody) at a dilution of 1:100,000 in 5%-TBS-T. The membrane was washed extensively before being developed using Enhanced Chemiluminesence (Pierce, SuperSignal West Pico). 54 CHARTER III - RESULTS 3.1 - Characterization of recombinant IDO The recombinant IDO protein obtained as described in the Material and Methods were characterized spectroscopically and kinetically against the values reported for the non recombinant human IDO. These results are indicated in Figure 14. The kinetics and spectroscopic parameters obtained are similar to the ones discussed previously in Table 3 and in the literature (Takikawa et al., 1988; Littlejohn et al., 2000). 3.2 - Detection and Identification of IDO inhibitors present in extracts of marine invertebrates A s described above, a library of 4500 extracts prepared from marine invertebrates by the Andersen laboratory was screened for the ability to inhibit purified recombinant IDO in vitro by means of a high throughput strategy. Although several extracts exhibited inhibitory activity, extracts from Garveia annulata and Xestospongia were particularly effective and were selected for further analysis. The results of work with these two extracts are presented below. 3.2.1 - Inhibitors from the pacific hydroid Garveia annulata The first of the extracts identified by high throughput screening assays (see Materials and Methods) to exhibit efficient inhibition of IDO was a methanol extract prepared from the Northwest Pacific hydroid Garveia annulata (Figure 15) that had been collected from the west coast of Vancouver Island. 55 Figure 14: Properties of recombinant human IDO: (A) L i n e w e a v e r - B u r k p l o t a n d k i n e t i c s p a r a m e t e r s o f r e c o m b i n a n t I D O i n K P B ( 1 0 0 m M , p H 6 . 5 ) , m e t h y l e n e b l u e ( 2 5 p M ) , b o v i n e l i v e r c a t a l a s e ( 2 0 0 p g / m L ) , a s c o r b i c a c i d ( 1 0 m M ) a n d 3 7 ° C , ( B ) s p e c t r a l o f I D O i n i t s r e d u c e d ( r e d ) a n d o x i d i z e d ( b l a c k ) f o r m ( K P B ( 1 0 0 m M , p H 6 . 5 ) a n d I D O ( 7 . 5 u M ) 56 Figure 15: Garveia annulata was collected at Barkley Sound (British Columbia). (A) D i v e r s c o l l e c t i n g s a m p l e s a t B a r k l e y S o u n d ; (B) c o l l e c t e d s a m p l e o f Garveia annulata. 5 7 Secondary metabolites from this organism have been studied previously by the Andersen group and have been shown to comprise a rich source of highly functionalized anthracene and naphthalene based polyketides (Fahy, et al, 1985; Fahy, et al., 1986a; Fahy, et al, 1986b, and Fahy, et al, 1987) that are the principal pigments of this organism. IDO assay-guided fractionation of this extract performed in collaboration with Alban Pereira in Professor Andersen's group led to the identification of the new polyketide annulin C (6) and the previously identified compounds annulin A (12) and annulin B (5) as the most potent IDO inhibitors present in this extract (Table 6). A second group of compounds from this extract were found to be somewhat less potent inhibitors of IDO. These inhibitors include the new compounds 2-hydroxygarveatin B (16) and garveatin E (19), as well as the previously reported compounds garveatin A (20), garveatin C (13), and 2-hydroxygarvin A (18). Garvin B (30), a secondary metabolite of G. annulata with a new carbon framework but lacking inhibitory activity, was also identified during the course of this investigation. 3.2.2 - Inhibitors from the sponges of the genus Xetospongia The other marine invertebrate source of efficient IDO inhibitors was a methanol extract obtained from Pacific sponges of the genus Xestospongia that were collected in Papua New Guinea by the Andersen group. Our IDO assay guided fractionation identified three quinones as strong IDO inhibitors: adociaquinones A (4) and B (1) and xestoquinone (7). 58 Table 6: Compounds identified by the in vitro assay as IDO inhibitors. [ p o t a s s i u m p h o s p h a t e b u f f e r ( 1 0 0 m M , p H 6 . 5 ) , m e t h y l e n e b l u e ( 2 5 p M ) , b o v i n e l i v e r c a t a l a s e ( 2 0 0 p g / m L ) , a s c o r b i c a c i d ( 1 0 m M ) , I D O ( 5 0 n M ) , 2 5 ° C ] C o m p o u n d s a r e l i s t e d i n t h e o r d e r o f t h e i r K \ . Number Formula Name SciFinder Registry number Empirical formula MW 1 O O H 25 nM Adocia-quinone B 113831-00-8 C 2 2 H 1 7 N 0 6 S 423.44 2 OH O 48 nM Juglone 481-39-0 Cio H 6 0 3 174.15 3 o 45 nM Dichlone 117-80-6 Cio H4 C12 O2 227.04 4 o o o ° 86 nM Adocia-quinone A 113830-99-2 C 2 2 H I 7 N 0 6 S 423.44 5 O O OH 123 nM Annulin B 105335-74-8 C21 H22 0 7 386.40 6 j? o — O OH ' o 144 nM Annulin C 873686-34-1 C2o H22 0 7 374.38 5 9 Cv i 7 o o 181 nM Xestoquinone 97743-96-9 C20 H) 4 O4 318.32 8 o 215 nM 2-Bromo 1,4-naphthho-quinone 2065-37-4 C l 0 H 5 Br 0 2 237.05 0 II 9 a} 0 334 nM 1 ^-naphtho-quinone 130-15-4 C10 H 6 O2 158.15 10 o 530 nM 2- methoxy-naphtho-quinone 2348-82-5 C n H 8 O3 188.18 o II 11 0 580 nM Vitamin K 3 Menadione 58-27-5 C „ H 8 0 2 172.18 \ J L O H i r 12 1 o 694 nM Annulin A 105335-73-7 C19 H20 O7 360.36 0 O OCH 3 0 \ II II 1 II 13 1.18 u M Garveatin C 99475-52-2 C22 H24 O5 368.42 O OH O C H 3 O 14 1.25 u M Garvalone C 109872-45-9 C 2 7 H 3 2 0 7 468.54 O OH OH 15 1.42 u M Garveatin F 109894-11-3 C2o H 2 2 0 5 342.39 6 0 16 O OH OH 1.42 uM 2-Hydroxy-garveatin B 109894-11-3 C2o H22 0 5 342.39 17 OH 1.8 uM 1 - naphthol 90-15-3 Cio Hjj O 144.17 18 O OH / 0 HO-V^><^ S Y^ T X ^ O C H 3 O ^ ^ ^ V ' ^ ^ ^ ^ ^ O C H a 2.3 (J.M 2-Hydroxy-garvin A 99457-97-3 C23 H 2 6 O7 414.45 19 O OH O 3.1 |xM Garveatin E 873536-71-1 C2o H2o 0 5 340.37 20 O OH OH O 3.2 uM Garveatin A 95388-04-8 C20 H2o O5 340.37 21 C& 3.4 uM 1 ^ -naphtho-quinone 524-42-5 Cio H 6 0 2 158.15 22 >32uM Warfarin 81-81-2 C19 H ) 6 O4 308.33 23 0 CH 3 CH 3 R— CH2 CH=C—(CH2 CH2 CH2 CH)3—CH3 >40uM Vitamin K] 84,80-0 C31 H 4 6 0 2 450.7 6 1 24 O OH o 42 u M Alizarin 72-48-0 C ] 4 H 8 0 4 240.21 25 N a S 0 4 » ^ / \ | /J \ ^ L ^ - / N a S O / ' ^^i^ § 0 4 N a -61 u M Sokostraterol sulfate 90352-20-8 C31 H 5 4 O 3 474.76 26 N a S O / ' ^ ^ N ^ § 0 4 N a -61 u M Halistanol trisulfate 79405-68-8 C29 H 5 2 0 3 448.72 27 1 H 100 u M Norharman 244-63-3 C„ H 8 N 2 168.19 28 o 100 u M Lawsone 83-72-7 C10 H 6 O3 174.15 29 c n 1.11 mM Coumarin 91-64-5 C9 H 6 O2 146.14 30 OH f ^ o -2.1 m M Garvin B 873686-42-1 C2o H 22 0 5 342.39 6 2 OH Dihydro-31 OH > 5 m M quinone 123-31-9 Cf, Hg O2 110.11 O OH / \ Ji JL _ ^ COO" 32 O C H 3 O OH O / ^ Y ^ N H 2 OH - 4 6 mM Doxorubicin 23214-92-8 C 2 7 H 2 9 N O „ 543.52 6 3 In addition to these compounds, the biossay-guided fractionation also led to identification of halistanol trisulfate ( 2 6 ) and sokotrasterol trisulfate ( 2 5 ) as D D O inhibitors present in several sponge extracts. Both compounds are relatively weak inhibitors of D D O , as indicated by A j^ values of ~ 60 uM. 3.2.3 - Structural Analogues of IDO Inhibitors obtained from marine invertebrates Knowing the structures of the inhibitors of new D D O inhibitors derived from natural products permitted evaluation of potential inhibitory properties of commercially available compounds with related structural elements. A variety of these compounds were evaluated by means of the high throughput assay strategy described above, and the resulting K\ values are provided in Table 6. 3.3 - Inhibition kinetics of 1-methyl-1, 4-naphthoquinone (menadione) Enzyme kinetics analyses were carried out as described in the Material and Methods section. As shown in Figure 16.A, E D O activities vary with the concentration of menadione added to the reaction mixture. The double reciprocal plot was a series of parallel lines indicating lower K m and decreasing V m a x . This result is characteristic of un-competitive inhibition (Figure 16.B). Lineweaver-Burk plots for different concentrations of an uncompetitive inhibitor yield a family of parallel lines that are diagnostic for uncompetitive inhibition. 64 B o * o -1 1— 30 60 [Trp] (MM) i 90 50 - I , , , 0 0.1 0.2 0.3 1/[trp] (1/uM) Figure 16: Inhibition of IDO by 1-methyl-1,4-naphthoquinone: (A) E f f e c t o f m e n a d i o n e o n t h e I D O a c t i v i t y ; (B) L i n e w e a v e r - B u r k p l o t ( 1 / V v e r s u s 1 / [ S ] ) [ p o t a s s i u m p h o s p h a t e b u f f e r ( 1 0 0 m M , p H 6 . 5 ) , m e t h y l e n e b l u e ( 2 5 p M ) , b o v i n e l i v e r c a t a l a s e ( 2 0 0 p g ) , a s c o r b i c a c i d ( 1 0 m M ) , I D O ( 5 0 n M ) a n d 2 5 ° C . M e n a d i o n e c o n c e n t r a t i o n s : 0 . 5 8 p M ( ) ; 1 . 1 7 p M ( • ) ; 1 . 7 6 p M ( x ) a n d z e r o ( • ) . 65 Thus, menadione is an uncompetitive inhibitor of human DDO in vitro. This type of inhibition is distinguished from "noncompetitive" inhibition, which affects only apparent V m a x , and from "competitive" inhibition, which affects only apparent Km. Using the equation (4) for uncompetitive inhibition (Segel, 1976), a K\ of 530 ± 50 nM was calculated. "max (I) = V m a x / ( 1 + [ ! ] / * , ) (4) Where V m a x is the maximal reaction rate with no menadione in the reaction mixture, [I] is the concentration of menadione in the reaction mixture and V m a x (I) is the apparent V m a x at a given [I]. 3.4 - Development of an assay for IDO activity in vivo Expression of a human gene in yeast can often produce a quantifiable phenotype, and screening for chemicals that reverse the phenotype has led to the identification of selective inhibitors of the protein expressed (Kurtz, et al., 1995; Perkins, et al., 2001; Simon and Bedalov, 2004). To provide an initial means by which the new DDO inhibitors discovered in this work could be evaluated in vivo, a yeast growth strategy of this type was implemented with the technical assistance of Aruna Balgi. Specifically, a single copy of the human DDO gene was introduced into the yeast strain EIS-20-2B at the LYS2 locus for expression controlled by the inducible GAL1 promoter. This yeast strain was selected for development of an in vivo assay of DDO activity because it lacks the major drug efflux pumps PDR5 and SNQ2 and, thus, is generally more sensitive to drugs that are added to the growth medium (Tugendreich, et al., 2001). A control strain, labeled CTRL, was 66 generated similarly by integrating the empty expression cassette. Expression of IDO by cells transformed with the IDO gene was confirmed by immunoblots. When grown in synthetic medium containing glucose to repress the GAL1 promoter, the CTRL and IDO-expressing strains of EIS-20-2B exhibited similar growth characteristics (Figure 17A). Culture growth was dependent on the presence of tryptophan in the medium because the EIS-20-2B strain lacks the TRP2 gene and is, consequently, a tryptophan auxotroph. A minimum of 20 u M tryptophan was required in the medium to support full growth. When incubated in galactose to induce expression from the GAL1 promoter, growth of CTRL cells exhibited a similar dependence on tryptophan concentration (Figure 17B). However, in the presence of galactose, cells expressing IDO grew poorly in medium containing less than 50 u M tryptophan, but they grew similarly to C T R L cells in the presence of high tryptophan concentrations (Figure 17B). These results indicated that human IDO expressed in yeast reduces tryptophan levels and consequently limits cell growth. Immunoblots demonstrate that the C T R L and IDO strains expressed no IDO under repressed conditions (Figure 17C) while under induced conditions, the strain transformed with the IDO gene exhibited a clear band with the size of full-length EDO while the CTRL strain did not. The expression of TDO fused to GFP was also monitored by fluorescence microscopy. The IDO-expressing cells exhibited no green fluorescence under growth conditions that IDO expression is repressed, but under induced growth conditions that IDO is expressed, they exhibited bright fluorescence, mainly in the cytoplasm (Figure 17D). Expression of the catalytically inactive IDO variant Ffis346Gly (Littlejohn, et al, 67 A — B — C T R L -GAL B ~ ~ C T R L + G A L — • — IDO -GAL — • — IDO +GAL Figure 17: Inhibition of yeast growth by human IDO. Y e a s t c e l l s b e a r i n g a n e m p t y e x p r e s s i o n c a s s e t t e ( C T R L ) o r t h e I D O e x p r e s s i o n c a s s e t t e ( I D O ) i n t e g r a t e d a t t h e LYS2 l o c u s w e r e g r o w n i n t h e p r e s e n c e o f g l u c o s e ( - G A L , ( A ) ) o r i n g a l a c t o s e ( + G A L , ( B ) ) a n d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f t r y p t o p h a n f o r 4 4 h . ( C ) , t h e s a m e s t r a i n s w e r e g r o w n i n t h e p r e s e n c e o f 1 0 0 u M T r p ( t o a l l o w g r o w t h o f t h e I D O + G A L c e l l s ) a n d I D O e x p r e s s i o n w a s m o n i t o r e d b y W e s t e r n b l o t t i n g . ( D ) , E x p r e s s i o n o f a G F P - I D O f u s i o n p r o t e i n i n t h e a b s e n c e a n d p r e s e n c e o f g a l a c t o s e w a s m o n i t o r e d b y f l u o r e s c e n c e m i c r o s c o p y ( F l u o r ) . 6 8 2003) caused little or no growth repression (Figure 23.B). Collectively, these results demonstrate that functional human IDO can be expressed conditionally in yeast, that IDO restricts yeast growth when tryptophan concentration in the growth medium is limited, and that growth repression requires catalytically active IDO. In medium containing galactose and Trp (25 u M ) , the IDO strain grew poorly, while the C T R L strain grew well (Figure 18). Under these conditions, selective IDO inhibitors would be expected to restore growth of the EDO strain without affecting the growth of the C T R L strain. EDO and C T R L cells grown in glucose were transferred to medium containing galactose and Trp (25 u M ) and distributed into 96-well plates. The cells were then exposed to various concentrations of M T H - T r p or 1-MT, and growth was quantified after 44 h at 30 °C. A t concentrations of 30 u M and greater, M T H - T r p partially restored the growth of the EDO strain (Figure 18A). The maximal level of growth restoration was about 25% at 100 u M M T H - T r p . O f note, M T H - T r p also inhibited the growth of the C T R L strain at 30 p M and greater (Figure 18 A ) . These results indicate that M T H - T r p inhibits recombinant EDO expressed in yeast, but that it is also toxic in the same concentration range, suggesting limited selectivity towards EDO. En contrast, 1MT did not cause growth restoration of the EDO strain at any concentration, and it was toxic to the C T R L strain at concentrations > 10 u M (Figure 18B), implying it does not inhibit recombinant EDO expressed in yeast or that it does so only at concentrations that are toxic. 69 A —e— CTRL +GAL B — © — CTRL +GAL — • — IDO +GAL — • — IDO +GAL Figure 18: Growth restoration as a drug-screening assay. C T R L a n d I D O c e l l s w e r e g r o w n i n t h e p r e s e n c e o f g a l a c t o s e a n d o f d i f f e r e n t c o n c e n t r a t i o n s o f ( A ) M T H - T r p o r ( B ) 1 - M T a n d g r o w t h w a s m o n i t o r e d a f t e r 4 4 h . ( C ) I D O c e l l s w e r e g r o w n i n t h e p r e s e n c e o f g a l a c t o s e o r o f p u r e c o m p o u n d s f r o m t h e N C I D i v e r s i t y S e t . G r o w t h r e s t o r a t i o n w a s m o n i t o r e d a t 4 4 h . T h e d i s t r i b u t i o n o f g r o w t h r e s t o r a t i o n a c t i v i t y o v e r t h e c o m p o u n d c o l l e c t i o n i s s h o w n . T h e i n s e t s s h o w t h e s a m e d a t a o n a d i f f e r e n t s c a l e t o h i g h l i g h t t h e s m a l l n u m b e r o f c o m p o u n d s e x h i b i t i n g s t r o n g g r o w t h r e s t o r a t i o n . ( D ) , a s ( C ) e x c e p t t h a t c e l l s w e r e t r e a t e d w i t h c r u d e e x t r a c t s f r o m m a r i n e s o u r c e s . 7 0 3.5 - Screening for in vivo inhibitors of IDO activity Having established the validity of the yeast growth assay for in vivo BOO activity, this new method was used to screen the 2500 purified reagents that comprise the NCI Diversity Set (Holbeck, 2004) and the Andersen library of crude extracts to evaluate their ability to restore growth of yeast expressing DDO. Again, these experiments were performed with the technical assistance of Aruna Balgi. The results obtained with the NCI Diversity Set are summarized in Fig. 18C. When added to the growth medium at a concentration of 2 uM, more than 80% of the compounds exhibited <10% growth restoration or <10% growth inhibition and were considered inactive. A small fraction (12%) exhibited >10% growth inhibition and were, therefore, toxic to yeast. Of the remaining compounds, 5% exhibited 10-40% growth restoration, 0.7% (17 compounds) showed 40-70% growth restoration and 0.4% (11 compounds) restored growth to 70-100% of the control rate. The activities of the 11 most effective compounds identified from the NCI Diversity Set were verified with a range of concentrations, as illustrated in Figure 18 for 1-MT and MTH-Trp. Two compounds produced false-positive readings because their precipitation in the yeast culture medium caused artifactual absorbance values. The EC50 (concentration that produces 50% of the maximal growth restoration for that compound) and the GI50 for yeast and for the NCI cancer cell panel for the nine confirmed positives are shown in Table 7. Their structures are shown in Figure 19. The EC50 for growth restoration ranged from 0.05 u M to 6 uM, consistent with their detection as active compounds at 2 p M in the screening assay. Most of these compounds exhibited little inhibition of yeast growth (GI50 >50 uM), except for NSC 31660. 71 NSC 67091 Figure 19: Compounds from the NCI Diversity Set that restore growth inhibited by IDO. S t r u c t u r a l f o r m u l a s a r e s h o w n i n t h e F i g u r e 7 2 Table 7: Effect of NCI Diversity Set compounds on IDO yeast growth restoration yeast and cancer cell growth inhibition. I C 5 0 f o r y e a s t g r o w t h r e s t o r a t i o n ( G R ) a n d t o x i c i t y t o y e a s t ( t o x ) a n d a v e r a g e G l 5 0 f o r t h e N C I c a n c e r c e l l p a n e l . N S C EC 5 o growth restoration (]iM)a GI50 yeast ( u M )b Average G I 5 0 N C I cancer cell panel (uM) b 31660 0.05 1 13 67091 1 100 52 372295 1 100 100 201863 2 >100 36 401366 3 60 114 116130 4 >100 nd c 99515 5 >100 35 117199 5 50 91 321578 6 >100 20 a Concentration that produces 50% of the maximal growth restoration for that compound b Concentration that causes 50% inhibition of cell growth (http://dtp.nci.nih.gov). c Not determined 73 The compounds show no obvious structural similarity to one another and span a range of structural classes. Only NSC 117199 contains a heterocycle resembling an indole. The ability to use this assay for screening crude extracts from natural sources was also analyzed. A collection of 875 methanol extracts from marine organisms was tested at 20 ug dried extract weight/mL. As shown in Figure 18D, 79% of the extracts were inactive in that they restored yeast growth less than 10%, or inhibited yeast growth less than 10%, whereas 2% inhibited yeast growth more than 10% and were, therefore, toxic to yeast. Of the remaining extracts, 17% restored growth by 10—40%, 1.7% (15 extracts) restored growth by 40-70% and 0.2% (2 extracts) restored yeast growth by 70-100%. One compound (NSC 401366, Fig. 20A) from the NCI Diversity Set was selected for further analysis. This compound was robust in restoring IDO-impaired growth at concentrations of 0.3-30 u M and was toxic to both CTRL and IDO strains at 100 u M (Fig. 20B). To determine whether NSC 401366 inhibits EDO directly, the effect of this compound on the steady-state activity of purified EDO was evaluated. These studies revealed that NSC 401366 inhibits EDO activity in vitro, and that this inhibition is overcome by increasing concentrations of tryptophan (Figure 20C). The apparent Km and V m a x values were determined from nonlinear least squares fitting. The enzyme V m a x remained constant as the concentration of inhibitor was increased, but the apparent Km increased linearly with increased inhibitor concentration (Figure 20D). Based on these data, the K{ for inhibition of EDO by NSC 401366 was determined to be 1.5 ± 0.2 uM. NSC 401366 is one of very few nonindolic EDO inhibitors (Gaspari, et al, 2006; Muller, et al, 2005). 74 Figure 20: Growth restoration and direct inhibition of IDO activity by NSC401366. (A) E f f e c t o f N S C 4 0 1 3 6 6 o n t h e g r o w t h o f C T R L a n d I D O s t r a i n s i n t h e p r e s e n c e o f g a l a c t o s e , c a r r i e d o u t a s i n F i g . 1 7 . (B) in vitro i n h i b i t i o n o f I D O a c t i v i t y b y N S C 4 0 1 3 6 6 . I D O a c t i v i t y w a s a s s a y e d a s d e s c r i b e d i n M e t h o d s i n t h e p r e s e n c e o f t h e f o l l o w i n g N S C 4 0 1 3 6 6 c o n c e n t r a t i o n s : 0 | i M ( O ) , 0 . 5 | i M ( • ) , 1 | i M ( • ) , 2 . 5 | i M ( • ) , a n d 5 u M ( A ) . ( C ) L i n e a r d e p e n d e n c e o f t h e a p p a r e n t Km o f I D O o n N S C 4 0 1 3 6 6 c o n c e n t r a t i o n . 75 3.6 - Effects of naphthoquinones on yeast cells expressing human IDO In view of the structures of IDO inhibitors identified from natural sources, several naphthoquinones commercially available were evaluated as potential IDO inhibitors. For example, the ability of one of these compounds, l-methyl-2,4-naphthoquinone (also known as menadione of vitamin K3) to restore yeast growth in the in vivo IDO assay described above is illustrated in Figure 21. Similar results were obtained with 2-methoxy-1,4-naphthoquinone and 2-bromo-l,4-naphthoquinone. Most of the other synthetic or natural occurring quinones were toxic to yeast or did not have any effect on the IDO expressing cell growth (data not shown). The results of similar assays performed with three other selected napththoquinones are shown in Figure 22. For these results, the abilities of these compounds to restore the growth of transformed yeast expression human IDO are reported as the growth restoration observed as calculated by the following relationship: % Growth restoration = (TEST - MED, D 0 ) / (CONT - MED, D 0 ) x 100 (5) Where TEST is Aeoonm of the well with IDO expressing cells with test compound, M E D I D O is the median value of A6oonm of the IDO expressing cells without the compound, and CONT is the median value of Aeoonm of empty vector-containing cells without the compound. 2-methoxy-l,4-naphthoquinone achieves maximal growth restoration (~ 80 %) at a concentration of 3 uM while Vitamin K3 exhibits a maximal growth restoration of 68% at a lower concentration (1 uM) and 2-bromo-l,4-naphthoquinone exhibits maximal growth recovery (~ 40 %) at 0.1 uM. 76 0.2 H 1 1—i—i—i—i i i i 1 1—i—i—i—i i i i 1 1—i—i—i—i i i i 1 10 100 1000 Vitamin K 3 concentration (nM) Figure 21: Effect of vitamin K 3,1-methyl-1,4-naphthoquinone, on the growth level of (•) human IDO expressing yeast and ( O ) CTRL. T h e i n i t i a l t r y p t o p h a n c o n c e n t r a t i o n w a s 1 0 0 p M . 7 7 IDO inhibitor concentration [nM] Figure 22: Growth restoration of human IDO expressing yeast by several naphthoquinones: C e l l s e x p r e s s i n g h u m a n I D O w e r e e x p o s e d t o v a r y i n g c o n c e n t r a t i o n s o f t h e I D O i n h i b i t o r ( O ) 2 - B r o m o - 1 , 4 - n a p h t h o q u i n o n e , ( • ) 2 -m e t h y l - 1 , 4 n a p h t h o q u i n o n e a n d ( • ) 2 - m e t h o x y - 1 , 4 n a p h t h o q u i n o n e . T h e p e r c e n t a g e o f g r o w t h r e s t o r a t i o n w a s a n a l y z e d a s i n d i c a t e d i n M a t e r i a l a n d M e t h o d s . P e r c e n t a g e o f g r o w t h r e s t o r a t i o n w a s c a l c u l a t e d a s d e s c r i b e d b y e q u a t i o n 5 . 7 8 3.7 - Cytochrome b5 and IDO The assay for IDO activity in vivo employs a yeast tryptophan auxotroph with a genomically integrated copy of the gene encoding human IDO while the current protocol involves growth of a yeast tryptophan auxotroph expressing EDO from a plasmid. Nevertheless, the growth of this yeast strain also exhibits a significantly greater dependence on the concentration of Trp in the medium than do control cells (Figure 23 A). Cultures of cells (100 \iM Trp) bearing the EDO gene or the empty vector exhibit a difference in OD600 of 0.493 ± 0.006 after 40 h growth. Expression of the inactive EDO variant has virtually no influence on yeast growth (Figure 23B), so the activity of the enzyme and not simply the expression of the protein is the basis for the effect of EDO expression on yeast growth observed in Figure 23 A. On the other hand, cultures of yeast lacking the gene encoding cytochrome bs exhibit a decrease in OD600 of just 0.12 ± 0.01 (at 100 uMTrp) when they also express EDO (Figure 24). En other words, removal of the cytochrome bs gene from the yeast genome reduced EDO activity by -75% as detected by this method. The amount of EDO expressed in these various cell lines as determined by Western blots was essentially identical (Figure 25), and EDO activity assays of yeast lysates (in the presence of excess ascorbate as a external reducing agent) produced identical results (data not shown). These findings establish that the intracellular concentrations of active EDO are essentially identical in both cell lines expressing this enzyme. While the occurrence of endogenous EDO or related activity in yeast is uncertain, our anti-serum against human EDO fails to cross-react with any yeast protein (Figure 25). Moreover, the growth characteristics of the control cultures lacking a gene encoding human EDO or transformed 79 with a plasmid bearing an inactive human IDO variant would account for any influence that an endogenous IDO activity might contribute to the current results. If cytochrome bs promotes the activity of IDO in vivo, then cytochrome bs reductase should exert a similar effect. To evaluate this possibility, the growth of yeast strains lacking either the mitochondrial form (MCR1) of cytochrome bs reductase (strain YKL150W) or the endoplasmic reticular form (CBR1) of the reductase (strain YIL043C) were studied (Fig. 25). The results of this experiment indicate that deletion of the gene encoding the mitochondrial reductase has a much smaller effect on the growth inhibition resulting from expression of IDO (Figure 26A) than does deletion of the gene encoding the endoplasmic reticular form of the reductase (Figure 26B). This differential effect of the two reductases is consistent with our previous observation that IDO expressed in yeast is located primarily in the yeast cytoplasm. Again, Western blot analysis (Figure 25) indicates that the intracellular concentration of IDO present in these yeast strains is comparable in all yeast strains studied in this work. 80 Figure 23: Growth rate analysis of yeast cultures expressing (A) wild-type IDO and (B) the His356Gly IDO variant in the presence of 2% galactose. Growth of yeast transformed with (•) an expression vector lacking and (•) possessing the gene encoding WT or the variant IDO are shown. 81 0.8 n [Trp] (MM) Figure 24: Growth rate inhibition cytochrome b5 deletion yeast mutant. Growth of yeast transformed with an expression vector lacking (•) and possessing (•) the gene encoding WT or the variant IDO are shown. 82 A B c D E • • P Figure 25: Western blot of the yeast strain lysates. (A) parental yeast cells with pARC25B, (B) parental Yeast cell + I D O , (C) cytochrome b 5 deletion mutant + I D O , (D) YKL150W deletion mutant with I D O , and (E) YIL043C deletion mutant with I D O . pARC25B is the empty vector control. 83 0.8 -I 0.8 -i 0.6 H O 0.4 O 0.2 -\ 250 500 750 [Trp] ( M M ) 1000 -i B 250 500 [Trp] (MM) 750 1000 Figure 26: Growth rate inhibition of cytochrome b 5 reductase deletion yeast mutant: (A) Y K L 1 5 0 w ( M C R 1 ) d e l e t i o n m u t a n t a n d (B) (•) Y I L 0 4 ( C B R 1 ) d e l e t i o n m u t a n t y e a s t c e l l s w i t h t h e I D O e x p r e s s i n g v e c t o r a n d ( • ) y e a s t t r a n s f o r m e d w i t h t h e s a m e v e c t o r l a c k i n g t h e g e n e f o r I D O w e r e g r o w n i n t h e p r e s e n c e o f 2 % g a l a c t o s e . 84 3 . 8 - Random mutagenesis of IDO As one means of identifying amino acid residues that are essential to the activity of IDO, a library of IDO variants was produced by PCR random mutagenesis and screened for catalytic activity. These mutant IDO genes were ligated into the pARC25B expression vector and were transformed into yeast cells as indicated in the Materials and Methods section. Individual colonies that contain a specific IDO variant were isolated by plating the transformed cells on selective media. Mutations that eliminate IDO activity in vivo were identified as described above by screening for yeast colonies that exhibited the growth characteristics shown in Figure 23 .B. The genes for several of the variants identified in this manner were analyzed (Table 8). Table 8 : Mutations that suppress IDO activity in vivo. K 1 3 6 T F 2 7 3 I S 3 9 8 P / R 2 9 6 S Q 3 4 8 P N 3 1 S Q 2 9 0 T e r m W 9 L / T 3 9 0 S M 2 9 9 L / Q 2 6 6 L E 2 5 4 V / E 2 7 5 D E 6 0 V / N 3 6 5 L D 7 4 N M 1 9 0 T / L 2 7 6 Q E 2 5 8 G / K 3 2 3 N L 3 7 N / N 2 7 4 D M 1 4 9 L V 8 2 D 85 From the variants that exhibited no activity in vivo, those that exhibited IDO activity in yeast lysates following addition of ascorbic acid were selected. One variant was identified that satisfied this criterion, the K136T IDO variant. The growth behavior of this variant is illustrated in Figure 27. This mutation is located in the middle of three consecutive lysyl residues found at positions 135, 136, and 137. To evaluate the functional contribution of this region of the protein, a series of IDO constructs were prepared in which these lysyl residues were replaced with glutaminyl residues. These variants are denoted K K K (wild type), QQQ, KQQ, QKQ and QQK, and their growth characteristics are shown in Figure 28. Cultures of cells (100 uM Trp) bearing the K K K TOO gene or the empty vector (CONT) exhibit a A O D 6 0 0 n m of 0.356 after 40 h growth (Figure 28) that represents 100% activity in vivo. Cultures of yeast carrying the QQQ variant exhibit a decrease in AOD 6 0 o n m of just 0.020 (at 100 u M Trp) with respect to CONT. This difference represents a 5% activity when these three lysines were replaced by glutamines. The results for the other variants are shown in Table 9. The amount of IDO expressed in these various cell lines as determined by Western blots was very close, and IDO activity assays of yeast lysates (in the presence of 10 mM ascorbate as an external reducing agent) produced identical results (Table 9). 86 250 500 750 1000 [Trp] (MM) Figure 27: Growth rate analysis of yeast cultures expressing the T136K IDO variant in the presence of 2% galactose. G r o w t h o f y e a s t t r a n s f o r m e d w i t h a n e x p r e s s i o n ( • ) l a c k i n g a n d ( • ) p o s s e s s i n g t h e g e n e e n c o d i n g t h i s v a r i a n t I D O a r e s h o w n . 87 Table 9: IDO in vivo and in vitro activity for IDO variants substituted at residues 135-137. R e s u l t s a r e d e r i v e d f r o m F i g u r e 2 8 . Positions IDO activity 135 136 137 In vivo (%) In vitro (%) K K K * 1 0 0 1 0 0 Q T Q ~ 0 - 9 9 Q Q Q - 5 - 9 6 Q Q K - 3 5 - 9 9 Q K Q - 4 0 - 9 7 K Q Q - 6 5 - 9 9 * W i l d - t y p e I D O 88 - • - C O N T - * - K K K Q Q Q - * - Q K Q - * - Q Q K - ^ K Q Q 250 500 750 1000 [Trp] ( u M ) Figure 28: Growth rate analysis of yeast cultures expressing KKK (wild-type IDO), QQQ, QKQ, KQQ, QQK variants and CONT (empty vector). 89 CHAPTER IV - DISCUSSION 4.1 - Overview The work presented in this dissertation was enabled by the development of a bacterial expression system for production of recombinant human TOO and a method for purification of the recombinant enzyme. At the time this work was started, one group had reported successful bacterial expression of IDO (Austin, et al, 2004; Littlejohn, et al., 2000), and during the course of this work, two other groups reported independent development of similar systems (Oda, et al., 2006; Papadbpoulou, et al., 2005; Sugimoto, et al., 2006). Although a systematic comparison of the efficiency of IDO production by the various systems is difficult to provide from the information that has been published, it appears that the system developed in the present work produces IDO in better yield than that provided by the system developed initially (Austin, et al., 2004; Littlejohn, et al, 2000) and comparable to those reported subsequently (Oda, et al., 2006; Papadopoulou, et al, 2005; Sugimoto, et al, 2006). The use of preparations derived from plants and other biological sources for therapeutic applications began in antiquity. During the Renaissance, the doctrine of signatures advanced by the Swiss botanist Paracelsus (Theophrastus Philippus Aureolus Bombastus von Hohenheim) argued that close observation of nature could lead to identification of herbs or trees near the site of origin of a disease that had curative capability. As reviewed by Jeffreys (2004), the discovery and development of acetylsalicylic acid (aspirin) as a pharmaceutical agent provides the first example of a modern therapeutic agent that developed from a natural source through the use of 90 chemical synthetic methods and rudimentary clinical trials, events that led to the first patent issued for a drug in 1898. The discovery of aspirin originated in the use of willow bark by the Reverend Edward Stone in the mid-eighteenth century for treatment of ague, a vague term referring primarily to malaria but including a wide range of unrelated conditions. Although Rev. Stone thought he had identified a convenient alternative to bark from the Peruvian cinchona tree used to treat the ague, the active agent in willow bark (salicylic acid) treats the symptoms of malaria rather than attacking the malaria parasite that causes the disease as does the quinine present in the bark of the cinchona tree. With the discovery that acetylation of salicylic greatly reduces gastric side effects, the development of a practical synthetic method by chemists at the Farbenfabriken vormals Friedrich Bayer & Company led to formation of a pharmaceutical division in a company that was previously focused solely on dye manufacturing. Today, the value of screening biological sources for natural products with specific biological activities is well recognized, and the field of pharmacognosy is an established discipline concerned with all aspects of drug development from such sources. Newman, et al. (2000) have reported that natural products play a major role in drug treatment, as over 50% of the most-prescribed drugs in the US have a natural product either as the drug or as "forebear" in the synthesis or design of the agent. Some of the more recently recognized sources of compounds with unusual structures and useful biological activities are marine invertebrates. The availability of a library of marine extracts assembled by Prof. Raymond Andersen's group at this University combined with the availability of facilities for high throughput activity 91 screening in the laboratory of Prof. Michel Roberge provided an excellent opportunity to search for new inhibitors of IDO with unanticipated structural characteristics and, potentially, with greater inhibitory capability than inhibitors described previously in the literature. From the results presented above, it is clear that these expectations have been met. Moreover, structural characterization of these compounds has led to the identification of a number of known, commercially available compounds as efficient IDO inhibitors. These new inhibitors combined with the three-dimensional structure of the enzyme that was determined by Sugimoto et al. (2006) towards the end of the present work, should enable informative, structure-based mechanistic studies of the IDO catalytic mechanism that were not previously possible. While the development of a high-throughput activity screen for IDO activity in vitro was essential to identification of these new inhibitors, the concomitant development of a high-throughput activity screen for IDO activity in vivo permitted identification of cytochrome bs as a possible electron transfer partner for IDO in vivo. Clearly, the availability of high throughput assays for IDO activity both in vivo and in vitro permits the design of a variety of informative experiments that identify a number of new directions for study in which physical (kinetic, thermodynamic and spectroscopic) methods can be employed to understand the structural and mechanistic bases for the catalytic and physiological activities of enzyme. 92 4.2 - Novel IDO inhibitors families In this research, several chemical families of agents exhibiting inhibition activities against human IDO have been discovered and characterized. Representative members of these chemical families are shown in Table 10. Brief discussions of each family are presented below. 4.2.1 - G. annulata family The most potent IDO inhibitors in this family are the new polyketide annulin C, and the known compounds annulin A and annulin B. No other biological role has been reported in the literature for this family of chemical compounds. While these compounds inhibit purified IDO in vitro, they did not promote in growth restoration in our in vivo yeast assay, presumably because they cannot cross the yeast plasma membrane due to the cell wall. 4.2.2 - Xestoquinone family Adociaquinones A (4) and B (1) and xestoquinone are among the strongest IDO inhibitors identified. Halenaquinone and xestoquinone (7) have been shown previously to be inhibitors of Cdc25B phosphatase (Cao, et al., 2005), phosphatidylinositol 3-kinase (Fujiwara, et al., 2001) and protein tyrosine kinase (Lee, et al., 1992). Both compounds also inhibit catalytic DNA unwinding and/or decatenation by topoisomerase II (Concepcion, et al., 1995). Xestoquinone (7) inhibits Pfnek-1, a novel protein kinase from the human malaria parasite Plasmodium falciparum and is a potential antimalarial agent (Laurent, et al., 2006). 93 Table 10: Novel IDO inhibitor families identified by several screening techniques. Family General Formula Range (uM) Type of inhibition against tryptophan N a p h t h o q u i n o n e s 0.48-100 Non-competitive G a r v e i a o OH 0.123-3.1 Non-competitive X e s t o q u i n o n e s o H i 0.025-0.181 Non-competitive N S C 4 0 1 3 6 6 NH NH N NH I H NHMe 1.5 Competitive P o l y h y d r o x y s t e r o i d s N a S ° 4 ' NaS04' S0 4Na -61 Non-competitive 9 4 This compound also induces apoptosis in PC 12 cells and causes calcium release through sulfhydryl modification of the skeletal sarcoplasmic reticulum (Ito, et al, 1999; Nakamura, et al, 2003; Sakamoto, et al, 1995). Even though these compounds are strong IDO inhibitors in vitro, adociaquinones A and B and xestoquinone do not inhibit IDO located in yeast cytoplasm, presumably because these compounds cannot cross the plasma membrane. Recent experiments in Prof. Anderson's laboratory have shown that by introducing functional groups into the xestoquinone structure to promote solubility in water, a promising inhibitory effect in yeast has been determined (-20% growth restoration). This work is currently in progress and suggests that this family of reagents may offer promise in development of a pharmacological agent for inhibition of TDO. 4.2.3 - Naphthoquinone family Upon considering the similarities among the various families of IDO inhibitors described in the previous sections, structurally related compounds that are commercially available were identified and evaluated as potential TDO inhibitors. From these experiments, an additional family of reagents comprised of small naphthoquinones was identified as potent IDO inhibitors. A few of these compounds are described below. Menadione, 2-methyl-l, 4-naphtoquinone (11), is a water-soluble derivative of vitamin Ki that is also referred to as vitamin K3. Menadione has been reported to inhibit the growth of several types of tumor cells (Chiou, et al, 1998; Tetef, et al, 1995; Verrax, et al, 2004). Menadione is reduced by NAD(P)H:quinone oxidoreductase (NQOR) in 95 vivo to form the hydroquinone at the expense of NAD(P)H (De Haan, et al., 2002; Hasspieler and Di Giulio, 1992), and the hydroquinone is oxidized by dioxygen to produce superoxide anion radical (O2"). The generation of superoxide by menadione is not responsible for IDO inhibition because the addition of large amounts of SOD to the reaction mixture does not affect this inhibition. Our results also showed that Menadione, 2-methoxy-2,4-naphthoquinones and 2-Bromo-2,4-Naphthoquinone (8) are able to cross the yeast plasma membrane and inhibit human IDO expressed in the yeast cytoplasm. Juglone (2), another member of this family, is found in the roots, bark and leaves of walnut trees (Inbaraj and Chignell, 2004; Varga, et al., 1996). Extracts derived from black walnut trees have been used in the treatment of acne, inflammatory disease and for hair dying (Inbaraj and Chignell, 2004), and juglone produces toxicosis in horses that chew the bark of walnut trees (True and Lowe, 1980). Juglone possesses antiviral, antibacterial and antifungal properties (Inbaraj and Chignell, 2004). There is some evidence that juglone is a mutagen (Tikkanen, et al., 1983) and carcinogen (Sugie, et ah, 1998), and it exhibited toxicity to yeast in the growth assays described above. Plumbagin (28), 5-hydroxy-2-methyl-1,4-naphthoquinone, is found in the root of Plumbago zeylanica L. This compound exhibits anticarcinogenic, anti-atherosclerotic and antimicrobial effects (Ding, et al., 2005; Hsieh, et al, 2005; Mossa, et al., 2004; Srinivas, et al., 2004). In addition, plumbagin exhibits an inhibitory effect on intestinal carcinogenesis causes cytogenetic and cell cycle changes in mouse Ehrlich ascites carcinoma, and possesses antiproliferation activity in human cervical cancer cells (Kini, et al., 1997; Srinivas, et al., 2004). As for juglone, plumbagin was toxic to yeast in the growth assays used in this study. 96 Doxorubicin (32) is a chemotherapeutic quinone used in treatment of many types of cancer either alone or in combination with other drugs. Nevertheless, the toxicity of Doxorubicin results in severe side-effects that are encountered frequently in the clinic (Lu, 2005). The cellular targets of doxorubicin are numerous and controversial (Keizer, et al., 1990; Ueno, et al., 2006). One target involves the stabilization of the topoisomerase II complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and thereby stopping the process of replication (Sun, et al., 2006). Even though doxorubicin possesses several structural similarities to IDO inhibitors discovered in the present work, it proved to be a very weak IDO inhibitor in vitro. It can be concluded that interesting IDO inhibitors for drug development occur in this family. However, more research is required to define the structural attributes required for these naphtoquinones to be strong and efficient inhibitiors of human EDO. 4.2.4-NSC 401366 family All of the new IDO inhibitors resulting from this work that have been described so far exhibit noncompetitive inhibition with respect to the substrate tryptophan. However, one compound that is competitive with respect to tryptophan was also identified. This compound, NSC 401366, was first identified in a somewhat different manner from the discovery of the other compounds in that it was first identified by the fact that it is robust in restoring IDO-impaired yeast growth at concentrations of 0.3 - 30 uM. 97 These studies revealed that NSC 401366 inhibits IDO activity and that this inhibition is overcome by increasing concentrations of tryptophan. The apparent Km and V m a x values determined from nonlinear fitting (see Results) resulted in a K\ for inhibition of IDO by NSC 401366 of 1.5 ± 0.2 uM. Thus, NSC 401366 is a more potent IDO inhibitor than other competitive inhibitors described in the literature, and one of a very small number of non-indolic competitive IDO inhibitors (Muller, et al., 2005). NSC 401366 is also structurally distinct from other IDO inhibitors as might be expected for a member of the NCI Diversity Set. NSC 401366 has been reported to be an inhibitor of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells (Kau et al, 2003) and to be an inhibitor of the protein phosphatase 2C by virtual screening (Rogers et al, 2006). 4.2.5 - Trisulfated polyhydroxysteroids family This family of inhibitors is represented by trisulfated polyhydroxysteroids, halistanol trisulfate (26) and sokotrasterol trisulfate (25). It was concluded that this family has a low pharmacological potential as IDO inhibitors because they have very low IDO inhibitory activities compared to the other families (ATjS ~ 60 uM) and because they present diverse biological activities previously discussed in the literature. For example, Halistanol trisulfate inhibits the enzyme endothelin convertin (Patil, et al, 1981), it has been shown to be cytoprotective against HIV (McKee, et al., 1994), and it is active in a thrombin receptor assay (Uchiyama, et al, 1992). Similarly, sokotrasterol trisulfate has been reported to be an angiogenesis-promoting steroid. By observing the changes in the relative abundances of over 1000 proteins in human endothelial cells treated with 98 sokotrasterol sulfate and vehicle-treated cells, it was concluded that this compound induces endothelial sprouting (Karsan, et al, 2005). These diverse biological activities reported in the literature indicate that this family of compounds has a low specificity towards a specific molecular target, presumably because they act as weak detergents. As a result, these compounds would have strong secondary effects if used for in vivo experiments and they have little potential for therapeutic development. 4 . 3 - The pharmacophore of human IDO Koehn and Carter (2005) have defined a pharmacophore as "the ensemble of steric and electronic features that is necessary to ensure optimal interactions with a specific biological target structure and to trigger (or to block) its biological response." Based on this concept and the structures of a few of the most potent inhibitors identified in the current work, the first pharmacophore for human IDO can be proposed (Figure 29). This structure is comprised of a naphthoquinone skeleton with some polar groups (usually oxygen) attached to the second ring. The determination of this pharmacophore was based on the similarities among the most potent inhibitors for IDO listed in Table 6. 99 Figure 29: Proposed Pharmacophore structure for human IDO. X = C o r S 100 4.4 - Mechanism of inhibition by menadione The kinetic results presented above establish that menadione inhibits human DDO by a mechanism that is uncompetitive with respect to tryptophan. Uncompetitive inhibition requires that the inhibitor affects the catalytic function of the enzyme but not substrate binding. Such inhibition is usually significant only for multi-substrate enzymes under conditions that the inhibitor competes with the substrate whose concentration is kept constant in the kinetics experiments (Voet, et al., 1998). Consequently, our findings suggest that menadione might be competing with dioxygen at the DDO active site. In an effort to identify a potential site for menadione binding in the DDO active site, the program AutoDock (Morris, et al., 1998; Solis and Wets, 1981) was used to simulate the interaction of menadione with the enzyme. AutoDock is a suite of automated software docking tools that predicts structural models for the binding of small molecules to receptors of known three-dimensional structure (de Graaf, et al., 2006; Mulakala, et al., 2006; Rogers, et al., 2006). With the guidance of Cecilia Chiu, initial models of the menadione-DDO complex produced by AutoDock 3.0 (Morris, et al, 1998) were adjusted manually with XtalView (McRee, 1999). The parameter files for AutoDock3.0 were prepared with the PRODRG server at Dundee (Schuttelkopf and van Aalten, 2004). The resulting model (Figure 30) indicates that menadione binds above the distal side of the heme in the hydrophobic active site. The binding environment is defined by Tyrl26, Phel63, Serl67, Phe226, Gly262, Ser263 and Ala264. The interaction of menadione with DDO is, thus, primarily through hydrophobic contacts with non-polar residues. This putative binding site is consistent with the kinetic results described above because binding of menadione in this manner places it some distance from the expected 101 A Figure 30: Proposed binding site for menadione at the active site of IDO as predicted by simulations generated with AutoDock as described in the text. ( A ) S t e r e o v i e w s o f t h e h e m e c a v i t y a n d ( B ) t o t a l p r o t e i n a r e s h o w n . A m i n o a c i d r e s i d u e s t h a t a r e l o c a t e d w i t h i n 7 A o f t h e b o u n d m e n a d i o n e a r e s h o w n i n h e m e c a v i t y . I n ( B ) , m e n a d i o n e i s s h o w n i n c y a n a n d h e m e i n r e d . 102 binding site for tryptophan (near Phe227 and Arg231). As a result, the binding of menadione at this site should not affect the binding of tryptophan significantly. At the same time, the binding of menadione at this site can be anticipated to have a significant influence on the interaction of dioxygen with the heme iron. The estimated free energy for formation of the IDO-menadione complex that is predicted by AutoDock is -9.13 kcal/mol, a value that corresponds to an estimated inhibition constant (Ki) of 2.0 x 10"7 M (200 nM) at 298.15 K. Notably, this value is the same order of magnitude as the experimentally determined K\ derived from kinetics experiments described above (580 nM at 298.15 K). Considering that Autodock treats the protein as a rigid body and permits torsional flexibility for the ligand only, this agreement is remarkable and perhaps fortuitous. Sugimoto et al. (2005) have indicated that Phe226 and Arg231 are involved in substrate recognition by hydrophobic interactions and by assuring the proper geometry for catalytic interaction of the substrate and dioxygen. The residues identified by Autodock as forming the menadione-binding site also merit comment. For example, Sugimoto et al. proposed that Ser263 interacts with the heme 7-propionate to stabilize the binding of heme to the apo-enzyme. This interaction could provide the basis for the reduction in activity of the Ser263Ala variant to 15% that of the wild-type enzyme. On the other hand, the Serl67Ala and Phel63Ala variants exhibit activity comparable to that of wild-type IDO. Thus, modeling result implies that substitutions for Phel63 and Ala264 may modify the inhibitory capability of menadione while having little or no effect on the dioxygenase activity of the enzyme. Future studies 103 of Ser263Gln or Ala264Ile mutants will provide more direct evidence to support this model. Westley and Westley (1996) have concluded that substrate-competitive inhibition may often be an inappropriate basis for design of potential therapeutic agents. Their analysis provided evidence that uncompetitive inhibitors are far more effective in open systems (constant input of substrates and removal of products) than are substrate-competitive inhibitors. As a result, menadione and its chemical family constitute a promising basis for the design of novel DDO inhibitors. Molecular modeling of the DDO-menadione complex in conjunction with pharmacological and NMR studies could help identify structure-activity relationships among naphthoquinone derivatives to facilitate design of a new generation of potent therapeutic agents with improved selectivity for DDO. 4.5 - IDO variants lacking activity in vivo As described above, several DDO variants produced by random mutagenesis resulted in enzyme lacking activity in vivo as detected by the yeast growth assay. The positions of amino acid substitutions identified in these inactive variants are indicated in Figure 31. Interestingly, these substitutions are distributed in a seemingly random fashion throughout the structure of the enzyme. Nevertheless, an interesting concentration of substitutions is apparent in the region of residues 273 to 276. Sugimoto et al. (2005) proposed catalysis requires optimal orientation of the substrate and dioxygen to permit proton abstraction, which results from the strict complementarity of the indole ring of the substrate and a hydrophobic group from the 104 Figure 31: Stereo figure of IDO Variants lacking activity in vivo. T h e p o s i t i o n s o f a m i n o a c i d s u b s t i t u t i o n s i d e n t i f i e d i n t h e y e a s t i n a c t i v e v a r i a n t s a r e i n d i c a t e d i n t h e f i g u r e . 105 protein. Specifically, these authors observed that although the Phe226Ala, Phe227Ala and Tyr231Ala variants do not exhibit an altered binding constant for tryptophan they do exhibit a dramatically reduced dioxygenase activity. Similarly, substitutions for residues 273 to 276 are also likely to alter the geometry required for tryptophan-dioxygen-heme interaction. Ultimately, characterization of variants at these positions is required to evaluate structural and functional roles of these residues. 4.6 - Strategies for evaluation of human enzyme function in vivo with yeast. Expression of a human gene in yeast can often produce a quantifiable phenotype, and screening for chemicals that reverse the phenotype has led to the identification of selective inhibitors of the protein expressed (Kurtz, et al., 1995; Simon and Bedalov, 2004; Tugendreich, et al., 2001) In this research, this technique was extended to an enzyme that degrades an essential component (tryptophan) required for yeast growth, and the effect of the titration of this essential component on the yeast growth was studied. It was established that this assay could be used for drug screening with crude extracts from natural sources and with pure chemicals. This capability is useful in drug discovery. In addition, this approach was used to identify a putative physiological redox partner for the recombinant enzyme. Expression of this foreign protein in selected yeast deletion mutants, allowed proteins involved in maintaining DDO in the active form in vivo to be identified. Several features of the yeast growth restoration assay make it appealing for drug screening. First, the system can in principle be used to express any monomelic protein target using a single set of plasmids. Second, inhibitors can be identified with a simple 106 and inexpensive absorbance measurement without addition of reagents to the test plates. Third, the growth restoration assay involves a positive selection, which means that compounds that are toxic to yeast will not give a false positive signal, a common problem with cellular assays that rely on inhibition of growth or the reduction of signal intensity. Finally, the assay combines attributes of cell-based screens, which' select for pharmaceutically-desirable properties such as the ability to cross cell membranes, with the specific protein targeting typically associated with in vitro assays. The present results also show that this technique can be used not only to search for novel inhibitors but also can be used to study the function of the enzyme in a cellular context. This technique can be applied to other metabolic enzyme that may be pharmacological targets in various human diseases. 4.7 - Cytochrome 6 5 and IDO interaction in yeast The present study demonstrates that human TDO expressed from a non-integrated yeast vector or from a cassette integrated into the genomic Lys2 locus of S. cereviseae is active in vivo and that a yeast tryptophan auxotroph expressing human IDO exhibits growth inhibition relative to identical yeast that lacks the human EDO gene. The ability to express active human IDO in yeast permitted development of strategies involving IDO expression in yeast knock-out mutants to evaluate physiological factors that may influence IDO activity. The reduction of sensitivity of yeast growth to the expression of active IDO resulting from lack of cytochrome 65 (Figure 32) and the endoplasmic cytochrome bs reductase provides compelling evidence that this cytochrome and its 1 0 7 can function in maintaining the activity of EDO in vivo. Notably, sensitivity of yeast growth to the expression of DDO was significantly less for the yeast mutant unable to produce the cytoplasmic reductase than was observed for yeast unable to produce the mitochondrial reductase. While the absence of the cytochrome or the reductase significantly reduces the growth inhibition resulting from EDO expression, absence of neither protein completely eliminates the growth effect of EDO. Presumably, in the absence of these proteins either the autoxidation of EDO occurs at a sufficiently slow rate that culture growth continues to be influenced by EDO to the extent observed or alternative sources of electrons remain that have not yet been identified. One alternative source of electrons could be NADPH-cytochrome P450 oxidoreductase. Disruption of the cytochrome bs gene generates no growth phenotype in a wild-type background but results in lethality when present in yeast also lacking NADPH-cytochrome P450 oxidoreductase. Thus, NADPH-cytochrome P450 oxidoreductase might compensate partially for the absence of cytochrome bs (Imai, 1981; Lamb, et al., 1999). In view of the preference of EDO for the superoxide anion radical relative to dioxygen as a substrate, another enzyme with potential consequences for activity of EDO in vivo is superoxide dismutase. As for NADPH-cytochrome P450 oxidoreductase, the functional consequences of eliminating superoxide dismutase on yeast physiology are sufficiently profound that the functional relationship between SOD and EDO in vivo will not be easily resolved through experiments of the type described here. Nevertheless, the present results provide the first indication that an electron donor for EDO may be present in vivo. The implied value of an enzymatic means of overcoming 109 the effects of autoxidation and maintaining EDO in the functional, reduced state as believed to occur with myoglobin and hemoglobin further supports the evolutionary relationship of IDO to the globin family (Suzuki, et al., 1998). Experimental and theoretical investigation of the interaction of cytochrome 65 with cytochrome c, myoglobin and hemoglobin over more than twenty years in Dr Mauk's laboratory has produced considerable insight into the manner in which these proteins recognize and bind to each other. In addition, cytochrome b$ is now known to be the physiological electron donor for a variety of metalloenzym.es (see Table 11 and 12). In these roles, cytochrome 65 may be an obligate component (O) or a modifier (M) for various enzymes. A modifier is an agent that binds to an enzyme and changes the final product of its enzymatic reaction. In either case, cytochrome bs is used as a component in many of the reactions, whether with NADPH-cytochrome P450 reductase (Table 11) or NADH-cytochrome bs reductase (Table 12), it appears to function as an obligate electron donor to the reaction. 110 Table 11: Cytochrome Independent enzyme systems with NADPH-cytochrome P450 reductase or NADH-cytochrome b5 reductase (from Schenkman and Jansson, 2003). R E D U C T A S E E N Z Y M E S U S T R A T E R O L E R E F E R E N C E Stearyl coenzyme A desaturase Stearyl coenzyme A 0 (Holloway and Katz, 1972; Oshino, et al., 1971; Shimakata, etal., 1972; Strittmatter, et al., 1974) A5-Desaturase 8,11,14-Eicosa-trienoic acid O * (Cho, et al., 1999a) A6-Desaturase Linoleic acid 0 * (Cho, et al., 1999b) NADPH-cytochrome P450 reductase Plasmalogen synthase 1-O-Alkyl phosphatidylethanol amine 0 (Paltauf, et al., 1974; Wykle,e/ al., 1972) or A7-Sterol 5 5-desaturase Cholest-7-en-3p-ol 0 (Reddy, et al., 1977) N A D H - cytochrome b5 reductase Cytochrome P 4 5 0 -11A1;C17,20 lyase, 17a-hydrolase 17a-OH-Progesterone, progesterone M (Ishii-Ohba, et al., 1984; Katagiri, et al., 1982; Shinzawa, et al., 1985) Methylsterol Monooxygenase 4,4-Dimethyl-5a-cholest 7-ene-3P-ol O (Fukushima, et al., 1981) C r v l - reductase C r V I 0 (Jannetto, et al., 2001) Metmyoglobin reductase Metmyoglobin 0 (Livingston, et al., 1985) Methemoglobin reductase Methemoglobin 0 (Goto-Tamura, et al., 1976; Hultquist, et al., 1974) M , m o d i f i e r i n t h e a b s e n c e o f N A D H - c y t o c h r o m e b 5 r e d u c t a s e ; O , o b l i g a t e r o l e , i.e., > 1 0 - f o l d e n h a n c e m e n t ; O * , p r o t e i n c o n t a i n s a c y t o c h r o m e 6 5 - d o m a i n . I l l Table 12: Cytochrome independent enzyme systems with NADPH-cytochrome P450 reductase (from Schenkman and Jansson, 2003). R E D U C T A S E E N Z Y M E S U S T R A T E R O L E R E F E R E N C E Cytochrome P 4 5 0 2B4 Methoxyflurane O (Canova-Davis, et al., 1985) Cytochrome P 4 5 0 2B1 /?-Nitrophenetole 0 (Kuwahara and Omura, 1980) Cytochrome P 4 5 0 2B4 Prostaglandin A ! Ei E 2 0 (Vatsis, et al., 1982) N A D P H -Cytochrome P 4 5 0 4B7 Arachindonic acid 0 (Loughran, et al., 2001) cytochrome P 4 5 0 reductase Cytochrome P 4 5 0 3 B6 /7-Nitroanisol 0 (Sugiyama, et al., 1982; Sugiyama, etal., 1980) Cytochrome P 4 5 0 3B4 Testoterone O (Guengerich, et al., 1986; Halvorson, et al., 1990; Imaoka, et al., 1992; Shet, et al., 1995) Multiple C Y P 1,2, 3, and 4 P450S Many substrate M (Schenkman and Jansson, 2003) M , m o d i f i e r i n t h e a b s e n c e o f N A D H - c y t o c h r o m e b5 r e d u c t a s e ; O , o b l i g a t e r o l e , i.e., > 1 0 - f o l d e n h a n c e m e n t 1 1 2 4.8 - Possible role of the residues 135,136 and 137 in IDO The results presented above indicate that Lysl35, 136 and 137 are required for IDO activity in vivo. However, substitution of these residues does not seem to affect IDO activity in vitro as measured in yeast lysates. These three consecutive lysines are located on the surface of the small domain and form part of one strand of a short p-sheet as illustrated in Figure 33. As can be seen in Figure 33, these lysyl residues are located in a flexible loop on the surface of the enzyme. One possible explanation for the influence of these residues on IDO activity as monitored by the yeast growth assays is that they may be involved in the interaction of EDO and cytochrome b$ in vivo. The involvement of lysyl residues on the surface of IDO in interaction of the enzyme with cytochrome bs is appealing at least in part because the cytochrome is an acidic protein (pi ~ 5.2) with a large, negatively charged electrostatic potential surface located on the region of the protein surface where one edge of the heme prosthetic group is exposed to solvent. The complexes formed by the cytochrome with other electron transfer proteins and metalloenzymes have generally been observed to involve considerable electrostatic stabilization through the interaction of this surface with positively charged electrostatic potential surfaces on the complementary protein. In the case of microsomal cytochromes P450, however, a role for the hydrophobic C-terminal membrane binding domain has also been proposed. Nevertheless, binding of cytochrome 65 to rat cytochrome P450 2B1 was inhibited (by 75%) by a synthetic peptide corresponding to P450 residues 116-134. Replacement of Lysl22 in this peptide, however, nearly abolished this inhibition (Omata, et al., 1994) 113 Figure 33: Location of Lys135,136 and 137 on the surface of IDO. T h e p r o x i m a l l i g a n d t o t h e h e m e i r o n ( H i s 3 4 6 ) i s a l s o s h o w n . 114 In erythrocytes, the reduction of methemoglobin is dependent upon direct electron transfer from ferrocytochrome bj. These two proteins form a complex that appears to involve complementary charge interactions between acidic groups of cytochrome b$ and basic groups of hemoglobin (Gacon, et al, 1980; Poulos and Mauk, 1983). Similar conclusions have been drawn from studies of the cytochrome 65-cytochrome c complex, in which four lysyl residues of cytochrome c have been proposed to interact with carboxylic groups surrounding the heme of cytochrome bs (Mauk et al, 1995; Northrup et al, 1993). It seems likely that the general mechanism involved in all these protein-protein interactions may also be is applicable to the interaction of cytochrome bs and IDO. 4.9 - Proposed molecular interaction between IDO and cytochrome bs To explore further the possible mechanism by which IDO and cytochrome bs may interact, initial modeling experiments have been attempted. While, many approaches to modeling complexes of electron transfer proteins of this type have been used in the thirty years following proposal of the first hypothetical structure of this type (Salemme, 1976), the current work used an internet-based program ClusPro (http://nrc.bu.edu/ciuster). This program performs rigid-body docking by evaluating a large number of putative complexes and retains a specified number of resulting complexes that exhibit favorable surface complementarities. A filtering method is then applied to this set of structures to rank those complexes with good electrostatic and desolvation free energies for further clustering (Comeau et al (2004); Chen et al (2003)). Twenty ranked, putative DDO-cytochrome bs complexes were generated in this manner. The complex exhibiting the lowest energy and involvement of three lysyl 115 residues interacting with residues on the surface of cytochrome bs was selected. Other putative complexes were not considered. Of the twenty ranked models, the eighth satisfied this condition (Figure 34). In this model, IDO lysyl residues 126 and 124 are in close contact with cytochrome glutamyl acid residues 54 and 57, respectively, (Figure 35) and presumably stabilize complex formation through formation of intermolecular hydrogen bonds. The role of lysyl 125 is not clear at present. This lysine may be required to force the neighboring lysines to adopt a position necessary for the formation of the complex due to charge repulsions. While this model provides an attractive explanation for the results obtained in the current work, rigorous experimental evaluation of the involvement of these residues in complex formation is, nevertheless, required. 4.10 - Future directions and concluding remark The results presented here constitute an initial effort to identify new small molecule inhibitors of human IDO from natural sources. The newly identified inhibitors described in this dissertation have potential use as biological tools and/or as lead compounds for drug discovery. If these inhibitors continue through the drug development process, efficacy and toxicity must be assessed in animal studies and clinical trials. Our inhibitors based on natural products provide a basis for future synthesis of new agents with improved pharmacokinetics and / or toxicology. 116 Figure 3 4 : Stereo views of the proposed model for the interaction of IDO and cytochrome b5 obtained by ClusPro. C y t o c h r o m e b5 i s s h o w n i n r e d ; I D O i s s h o w n i n b l a c k . A r r o w s i n d i c a t e t h e p o s i t i o n o f t h e t h r e e c o n s e c u t i v e l y s y l r e s i d u e s i n I D O . 1 1 7 Cytochrome b5 Figure 3 5 : Two different views of the proposed interaction between IDO lysyl residues (yellow) and cytochrome glutamyl residues (blue). C l o s e - u p o f t h e c o m p l e x s h o w n i n F i g u r e 3 4 . 1 1 8 Use of compounds identified by screening of natural products as synthetic lead compounds for combinatorial approaches or as sources of simplified molecules is exemplified by work with bryostatins, ecteinascidins, and flavopiridiol, biologically active compounds that have been designed and synthesized based on knowledge gained from studies of natural products. Future synthetic worked based on the inhibitors described in this work may lead to a similar course of drug development for IDO inhibitors. The structural basis for the interaction of the IDO inhibitor discovered in the present work remains undefined. Thus, one direction for further development of this work is to identify the mechanisms by which these inhibitors bind to and inhibit IDO. One approach to achieving this goal is to prepare crystals of IDO-inhibitor complexes for structural characterization. Although the structure of IDO has been determined by this method, however, the resolution that has been achieved is limited, and preparation of suitable crystals remains challenging. Thus, the alternative of characterizing IDO-inhibitor interactions by NMR methods is highly attractive as demonstrated in related studies with other proteins (Krishna, et al, 1999; London, 1999; Ni, 1994; So, et al, 2003; Ruan, et al, 2005). At present, no NMR studies of IDO have been reported, so considerable effort will be required for successful use of this strategy as well. The presence of Fe(JJI)-heme at the active site of IDO provides an additional challenge to the use of NMR spectroscopy owing to the paramagnetic character of the enzyme in this oxidation state. Reduction to Fe(U)-heme reduces this problem, but even in this form heme ring currents can be expected to influence the spectroscopic properties of the protein, and maintenance of the reduced state adds the complication of manipulating the 119 protein under anaerobic conditions. Nevertheless, NMR spectroscopy promises to be a highly informative means of characterizing IDO-inhibitor complexes and the structural dynamics of the active site of IDO. The thermodynamics of IDO-inhibitor complex formation also merit study. The method of choice for such studies is isothermal titration calorimetry, a method that would provide both the stability constants for IDO-inhibitor complex formation and the enthalpy of this interaction. Recent studies with tartrate dehydrogenase are just one example of many in which this approach has provided such information (Karsten and Cook, 2006). In addition to these studies with wild-type IDO, the variants of the enzyme should be designed on the basis of the hypothetical structure for the IDO-menadione complex that was developed by molecular modeling in the present work. Specifically, Ser263Gln and Ala264Ile TDO variants could be prepared and studied for their susceptibility to inhibition by menadione to see if they participate in binding of this inhibitor. Those variants that exhibit altered response to menadione should be investigated further by the NMR and calorimetric methods described above. The interaction of IDO and cytochrome bs should be characterized by kinetic, thermodynamic, and structural methods. For example, the kinetics of electron transfer from ferricytochrome 65 to Fe(m)-IDO could be studied by anaerobic stop-flow spectroscopy as previously reported for the reduction of ferricytochrome c by ferricytochrome £5 (Eltis, et al, 1991). Thermodynamic properties of complex formation could, again, be studied by isothermal titration calorimetry or by potentiometric titration (Mauk, et al, 1991; Mauk, et al, 1994). In view of the highly limited success in crystallizing related pairs of electron transfer proteins for structural characterization, an 120 approach to gaining structural insight concerning IDO-cytochrome b5 interaction that is more likely to succeed is, again, provided by NMR spectroscopy (Hartshorn, et al, 1987; Moore, et al, 1998; Burch, et al, 1990; Horn, et al, 2000; Shao, et al, 2003; Deep, et al, 2005) could be provided. In addition, the crystallization of the complex between these two heme proteins might be attempted. These studies will indicate which amino acid residues in both proteins are required for recognition and interaction of these two physiological partners. 121 REFERENCES Alani, E. , Cao, L. and Kleckner, N. (1987). A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 116, 541-5 Arciero, D. M . and Lipscomb, J. D. (1986). Binding of nO-labeled substrate and inhibitors to protocatechuate 4,5-dioxygenase-nitrosyl complex. Evidence for direct substrate binding to the active site Fe 2 + of extradiol dioxygenases. J Biol Chem 261,2170-8. Austin, C. J., Mizdrak, J., Matin, A., Sirijovski, N., Kosim-Satyaputra, P., Willows, R. D., Roberts, T. H. , Truscott, R. J., Polekhina, G., Parker, M . W. and Jamie, J. F. (2004). Optimised expression and purification of recombinant human indoleamine 2,3-dioxygenase. Protein Expr Purifil, 392-8. Barcelo-Batllori, S., Andre, M . , Servis, C , Levy, N., Takikawa, O., Michetti, P., Reymond, M . and Felley-Bosco, E. (2002). Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases. Proteomics 2, 551-60. Bianchi, M . , Bertini, R. and Ghezzi, P. (1988). Induction of indoleamine dioxygenase by interferon in mice: a study with different recombinant interferons and various cytokines. Biochem Biophys Res Commun 152,237-42. Burch, A . M . , Rigby, S.E., Funk, W.D., MacGillivray, R.T., Mauk, M.R., Mauk, A . G . and Moore, G.R. (1990) NMR characterization of surface interactions in the cytochrome ^-cytochrome c complex. Science 247, 831-3. Burke, D., Dawson, D. and Stearns, T. (2000). Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press. Cady, S. G. and Sono, M . (1991). 1-Methyl-DL-tryptophan, p-(3-benzofuranyl)-DL-alanine (the oxygen analog of tryptophan), and P -[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of tryptophan) are competitive inhibitors for indoleamine 2,3-dioxygenase. Arch Biochem Biophys 291, 326-33. Canova-Davis, E. , Chiang, J. Y. and Waskell, L. (1985). Obligatory role of cytochrome bs in the microsomal metabolism of methoxyflurane. Biochem Pharmacol 34, 1907-12 122 Cao, S., Foster, C , Brisson, M . , Lazo, J. S. and Kingston, D. G. (2005). Halenaquinone and xestoquinone derivatives, inhibitors of Cdc25B phosphatase from a Xestospongia sp. Bioorg Med Chem 13, 999-1003. Chen R., Li L. and Weng Z.(2003) ZDOCK: An initial-stage protein docking algorithm. proteins 52, 80-7. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G. and Thompson, J. D. (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31, 3497-500. Chiou, T. J., Chou, Y. T. and Tzeng, W. F. (1998). Menadione-induced cell degeneration is related to lipid peroxidation in human cancer cells. Proc Natl Sci Counc Repub China B 22, 13-21. Cho, H. P., Nakamura, M . and Clarke, S. D. (1999a). Cloning, expression, and fatty acid regulation of the human delta-5 desaturase. J Biol Chem 274, 37335-9. Cho, H. P., Nakamura, M . T. and Clarke, S. D. (1999b). Cloning, expression, and nutritional regulation of the mammalian Delta-6 desaturase. J Biol Chem 21 A, 471-7. Comeau, S.R., Gatchell, D.W., Vajda, S., and Camacho, C.J. (2004) ClusPro: an automated docking and discrimination method for the prediction of protein complexes. Bioinformatics 20, 45-50 Concepcion, G. P., Foderaro, T. A., Eldredge, G. S., Lobkovsky, E. , Clardy, J., Barrows, L. R. and Ireland, C. M . (1995). Topoisomerase U-mediated DNA cleavage by adocia- and xestoquinones from the Philippine sponge Xestospongia sp. J Med Chem 38,4503-7. Cristea, M . , Osbourn, A. E. and Oliw, E. H. (2003). Linoleate diol synthase of the rice blast fungus Magnaporthe grisea. Lipids 38, 1275-80. Deep, S., Im, S.C., Zuiderweg, E.R. and Waskell, L. (2005) Characterization and calculation of a cytochrome c-cytochrome bs complex using NMR data. Biochemistry 44, 10654-68. De Leon, I. P., Sanz, A., Hamberg, M . and Castresana, C. (2002). Involvement of the Arabidopsis alpha-DOXl fatty acid dioxygenase in protection against oxidative stress and cell death. Plant J 29, 61-2. De Felippis, M . R., Murthy, C. P., Faraggi, M . and Klapper, M . H . (1989). Pulse radiolytic measurement of redox potentials: the tyrosine and tryptophan radicals. Biochemistry 28,4847-53. 123 De Graaf, C , Oostenbrink, C , Keizers, P. H., van der Wijst, T., Jongejan, A. and Vermeulen, N. P. (2006). Catalytic site prediction and virtual screening of cytochrome P450 2D6 substrates by consideration of water and rescoring in automated docking. J Med Chem 49, 2417-30. De Haan, L. H. , Boerboom, A. M . , Rietjens, I. M . , van Capelle, D., De Ruijter, A. J., Jaiswal, A. K. and Aarts, J. M . (2002). A physiological threshold for protection against menadione toxicity by human NAD(P)H:quinone oxidoreductase (NQOl) in Chinese hamster ovary (CHO) cells. Biochem Pharmacol 64, 1597-603. Ding, Y. , Chen, Z. J., Liu, S., Che, D., Vetter, M . and Chang, C. H. (2005). Inhibition of Nox-4 activity by plumbagin, a plant-derived bioactive naphthoquinone. J Pharm Pharmacol57, 111-6. Edgell, C.J., McDonald, C.C. and Graham, J.B. (1983) Permanent cell line expressing human factor VHI-related antigen established by hybridization, Proc. Natl. Acad. Sci. USA 80. 3734-37. Eguchi, N., Watanabe, Y., Kawanishi, K., Hashimoto, Y. and Hayaishi, O. (1984). Inhibition of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase by beta-carboline and indole derivatives. Arch Biochem Biophys 2 3 2 , 602-9. Fahy, E.; Andersen, R.J.; Cun-heng, H.; Clardy, J. (1985) Garveatin A, an antimicrobial l(4H)-anthracenone derivative from the hydroid Garveia annulata. J Org Chem 50, 1149-1150. Fahy, E.; Andersen, R.J.; Van Duyne, G.D., H.; Clardy, J. (1986a). Metabolites of the marine hydroid Garveia annulata: garveatins B and C, 2-hydroxygarvin A, and garvin A quinone. / Org Chem 51,57-61. Fahy, E.; Andersen, R.J.; Xu, C ; Clardy, J. (1986b). Annulins A and B, metabolites of the marine hydroid Garveia annulata. J Org Chem 51, 5145-8. Fahy, E.; Andersen, R.J. (1987). Minor metabolites of the marine hydroid Garveia annulata. J Can Chem 65, 376-83 Frumento, G., Rotondo, R., Tonetti, M . , Damonte, G., Benatti, U. and Ferrara, G. B. (2002). Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase. J Exp Med 196,459-68. Fujigaki, H. , Saito, K., Lin, F., Fujigaki, S., Takahashi, K., Martin, B. M . , Chen, C. Y., Masuda, J., Kowalak, J., Takikawa, O., Seishima, M . and Markey, S. P. (2006). Nitration and inactivation of IDO by peroxynitrite. J Immunol 176, 372-9. 124 Fujiwara, FL, Matsunaga, K., Saito, M . , Hagiya, S., Furukawa, K., Nakamura, H. and Ohizumi, Y. (2001). Halenaquinone, a novel phosphatidylinositol 3-kinase inhibitor from a marine sponge, induces apoptosis in PC 12 cells. Eur J Pharmacol 413, 37-45. Fukushima, FL, Grinstead, G. F. and Gaylor, J. L. (1981). Total enzymic synthesis of cholesterol from lanosterol. Cytochrome 65-dependence of 4-methyl sterol oxidase. J Biol Chem 256,4822-6. Gacon, G., Lostanlen, D., Labie, D. and Kaplan, J. C. (1980). Interaction between cytochrome bs and hemoglobin: involvement of beta 66 (E10) and beta 95 (FG2) lysyl residues of hemoglobin. Proc Natl AcadSci U SA 77, 1917-21. Gaspari, P., Banerjee, T., Malachowski, W. P., Muller, A. J., Prendergast, G. C , DuHadaway, J., Bennett, S. and Donovan, A. M . (2006). Structure-activity study of brassinin derivatives as indoleamine 2,3-dioxygenase inhibitors. J Med Chem 49, 684-92. Goto-Tamura, R., Takesue, Y. and Takesue, S. (1976). Immunological similarity between NADH-cytochrome bs reductase of erythrocytes and liver microsomes. Biochim Biophys Acta 423,293-302. Grohmann, U. , Fallarino, F. and Puccetti, P. (2003). Tolerance, DCs and tryptophan: much ado about IDO. Trends Immunol 24,242-8. Guengerich, F. P., Distlerath, L. M . , Reilly, P. E. , Wolff, T., Shimada, T., Umbenhauer, D. R. and Martin, M . V. (1986). Human-liver cytochromes P-450 involved in polymorphisms of drug oxidation. Xenobiotica 16, 367-78. Halvorson, M . , Greenway, D., Eberhart, D., Fitzgerald, K. and Parkinson, A. (1990). Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IHA1). Arch Biochem Biophys 277, 166-80. Hartshorn, R.T., Mauk, A.G. , Mauk, M.R. and Moore, G.R. (1987) NMR study of the interaction between cytochrome bs and cytochrome c. Observation of a ternary complex formed by the two proteins and [Cr(en)3]3+. FEBSLett. 213, 391-5. Hasspieler, B. M . and Di Giulio, R. T. (1992). DT diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase] facilitates redox cycling of menadione in channel catfish (Ictalurus punctatus) cytosol. Toxicol Appl Pharmacol 114, 156-61. Hayaishi, O. (1976). Properties and function of indoleamine 2,3-dioxygenase. J Biochem (Tokyo) 19, 13-21. 125 Hayaishi, O., Hirata, F., Ohnishi, T., Henry, J. P., Rosenthal, I. and Katoh, A. (1977). Indoleamine 2,3-dioxygenase: incorporation of 1802~ and 18C»2 into the reaction products. JBiol Chem 252,3548-50. Hayaishi, O., Katagiri, M . and Rothberg, S. (1955). Mechanism of the pyrocatechase reaction. / . Am. Chem. Soc. 77, 5450-1. Hayaishi, O., Rothberg, S., Mehler, A. H. and Saito, Y. (1957). Studies on oxygenases; enzymatic formation of kynurenine from tryptophan. JBiol Chem 229, 889-96. Hirata, F., Ohnishi, T. and Hayaishi, O. (1977). Indoleamine 2,3-dioxygenase. Characterization and properties of enzyme. O2" complex. JBiol Chem 252, 4637-42. Holbeck, S. L. (2004). Update on NCI in vitro drug screen utilities. Eur J Cancer 4 0 , 785-93. Holloway, P. W. and Katz, J. T. (1972). A requirement for cytochrome bs in microsomal stearyl coenzyme A desaturation. Biochemistry 11, 3689-96. Horn, K., Ma, Q . F . , Wolfe, G., Zhang, H. , Storch, E .M. , Daggett, V. , Basus, V.J. and Waskell, L. (2000) NMR studies of the association of cytochrome 65 with cytochrome c. Biochemistry 39,14025-39. Hsieh, Y. J., Lin, L. C. and Tsai, T. H. (2005). Determination and identification of plumbagin from the roots of Plumbago zeylanica L. by liquid chromatography with tandem mass spectrometry. J Chromatogr A 1083, 141-5. Hultquist, D. E. , Dean, R. T. and Douglas, R. H. (1974). Homogeneous cytochrome bs from human erythrocytes. Biochem Biophys Res Commun 6 0 , 2 8 - 3 4 . Hwu, P., Du, M . X., Lapointe, R., Do, M . , Taylor, M . W. and Young, H. A. (2000). Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. J Immunol 164, 3596-9. Imai, Y. (1981). The roles of cytochrome bs in reconstituted monooxygenase systems containing various forms of hepatic microsomal cytochrome P-450. J. Biochem. (Tokyo) 89, 351-62. Imaoka, S., Imai, Y., Shimada, T. and Funae, Y. (1992). Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity. Biochemistry 31, 6063-9. Inbaraj, J. J. and Chignell, C. F. (2004). Cytotoxic action of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes. Chem Res Toxicol 17, 55-62. 126 Ishii-Ohba, H. , Matsumura, R., Inano, H. and Tamaoki, B. (1984). Contribution of cytochrome bs to androgen synthesis in rat testicular microsomes. J Biochem (Tokyo) 95, 335-43. Ito, M . , Hirata, Y. , Nakamura, H. and Ohizumi, Y. (1999). Xestoquinone, isolated from sea sponge, causes C a 2 + release through sulfhydryl modification from skeletal muscle sarcoplasmic reticulum. J Pharmacol Exp Ther 291, 976-81 Jannetto, P. J., Antholine, W. E. and Myers, C. R. (2001). Cytochrome bs plays a key role in human microsomal chromium(VI) reduction. Toxicology 159, 119-33. Jeffreys, D. (2004). Aspirin. The Remarkable Story of a Wonder Drug. Bloomsbury. Kau, T.R., Schroeder, F., Ramaswamy, S., Wojciechowski, C. L. , Zhao, J. J., Roberts, T. M . , Clardy, J., Sellers, W. R. and Silver, P. A. (2003). A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells. Cancer Cell 4, 463-76. Karsten, W. E. and Cook, P. F. (2006) An isothermal titration calorimetry study of the binding of substrates and ligands to the tartrate dehydrogenase from Pseudomonas putida reveals half-of-the-sites reactivity. Biochemistry 45, 9000-6 Katagiri, M . , Suhara, K., Shiroo, M . and Fujimura, Y. (1982). Role of cytochrome bs in the cytochrome P-450-mediated C21 - steroid 17, 20 - lyase reaction. Biochem Biophys Res Commun 108, 379-84. Kawamichi, H. and Suzuki, T. (1998). The cDNA-derived amino acid sequence of indoleamine dioxygenase like-myoglobin from the gastropod mollusc Omphalius pfeifferi. J Protein Chem 17, 651-6. Keizer, H. G., Pinedo, H. M . , Schuurhuis, G. J. and Joenje, H. (1990). Doxorubicin (adriamycin): a critical review of free radical-dependent mechanisms of cytotoxicity. Pharmacol Ther 47,219-31. Kini, D. P., Pandey, S., Shenoy, B. D., Singh, U. V. , Udupa, N., Umadevi, P., Kamath, R., Nagarajkumari and Ramanarayan, K. (1997). Antitumor and antifertility activities of plumbagin controlled release formulations. Indian J Exp Biol 35, 374-9. Kobayashi, K., Hayashi, K. and Sono, M . (1989). Effects of tryptophan and pH on the kinetics of superoxide radical binding to indoleamine 2,3-dioxygenase studied by pulse radiolysis. JBiol Chem 264, 15280-3. Korlimbinis, A., Hains, P. G., Truscott, R. J. and Aquilina, J. A. (2006). 3-Hydroxykynurenine oxidizes a-crystallin: potential role in cataractogenesis. Biochemistry 45, 1852-60. 127 Koehn, F. E. and Carter, G. T. (2005). The evolving role of natural products in drug discovery. Nat Rev Drug Discov 4,206-20 Krishna, N.R. and Moseley, H.N B. (1999) in: N.R. Krishna,, L.J. Berliner (Eds.), Structural computation and dynamics in protein NMR, vol. 17, Kluwer Academic, New York, pp. 223-307. Kulmacz, R. J. and Lands, W. E . (1984). Prostaglandin H synthase. Stoichiometry of heme cofactor. J Biol Chem 259, 6358-63. Kurtz, S., Luo, G., Hahnenberger, K. M . , Brooks, C , Gecha, O., Ingalls, K., Numata, K. and Krystal, M . (1995). Growth impairment resulting from expression of influenza virus M2 protein in Saccharomyces cerevisiae: identification of a novel inhibitor of influenza virus. Antimicrob Agents Chemother 39,2204-9. Kuwahara, S. and Omura, T. (1980). Different requirement for cytochrome bs in NADPH-supported O-deethylation of p-nitrophenetole catalyzed by two types of microsomal cytochrome P-450. Biochem Biophys Res Commun 9 6 , 1562-8. Lamb, D. C , Kelly, D. E. , Manning, N. J., Kaderbhai, M . A. and Kelly, S. L. (1999). Biodiversity of the P450 catalytic cycle: yeast cytochrome 65/NADH cytochrome bs reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction. FEBS Lett. 4 6 2 , 2 8 3 - 2 8 8 . Laurent, D., Jullian, V. , Parenty, A., Knibiehler, M . , Dorin, D., Schmitt, S., Lozach, O., Lebouvier, N., Frostin, M . , Alby, F., Maurel, S., Doerig, C , Meijer, L. and Sauvain, M . (2006). Antimalarial potential of xestoquinone, a protein kinase inhibitor isolated from a Vanuatu marine sponge Xestospongia sp. Bioorg Med Chem 14,4477-82. Lee, R. H. , Slate, D. L. , Moretti, R., Alvi, K. A. and Crews, P. (1992). Marine sponge polyketide inhibitors of protein tyrosine kinase. Biochem Biophys Res Commun 184, 765-72. Leeds, J. M . ; Brown, P. J.; McGeehan, G. M. ; Brown, F. K.; Wiseman, J. S. (1993) Isotope effects and alternative substrate reactivities for tryptophan 2,3-dioxygenase. J Biol Chem 268, 17781-6. Littlejohn, T. K., Takikawa, O., Skylas, D., Jamie, J. F., Walker, M . J. and Truscott, R. J. (2000). Expression and purification of recombinant human indoleamine 2, 3-dioxygenase. Protein Expr Purif 19, 22-9. Littlejohn, T. K., Takikawa, O., Truscott, R. J. and Walker, M . J. (2003). Asp274 and His346 are essential for heme binding and catalytic function of human indoleamine 2,3-dioxygenase. JBiol Chem 278,29525-31. 128 Liu, W., Rogge, C. E. , Bambai, B., Palmer, G., Tsai, A. L. and Kulmacz, R. J. (2004). Characterization of the heme environment in Arabidopsis thaliana fatty acid a-dioxygenase-1. J Biol Chem 279,29805-15. Livingston, D. J., McLachlan, S. J., La Mar, G. N. and Brown, W. D. (1985). Myoglobin: cytochrome bs interactions and the kinetic mechanism of metmyoglobin reductase. JBiol Chem 260, 15699-707. • London, R.E. (1999). Theoretical analysis of the inter-ligand overhauser effect: a new approach for mapping structural relationships of macromolecular ligands. J. Magn. Reson 141, 301-11. Lou, B. S., Snyder, J. K., Marshall, P., Wang, J. S., Wu, G., Kulmacz, R. J., Tsai, A. L. and Wang, J. (2000). Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase. Biochemistry 39, 12424-34. Loughran, P. A., Roman, L. J., Miller, R. T. and Masters, B. S. (2001). The kinetic and spectral characterization of the E. co//-expressed mammalian CYP4A7: cytochrome bs effects vary with substrate. Arch Biochem Biophys 385, 311-21. Lu, P. (2005). Monitoring cardiac function in patients receiving doxorubicin. Semin Nucl Med 35, 197-201. Maezono, K., Tashiro, K. and Nakamura, T. (1990). Deduced primary structure of rat tryptophan-2,3-dioxygenase. Biochem Biophys Res Commun 170, 176-81. Mason, H. S., Fowlks, W. L. and Peterson, E. (1955). Oxygen transfer and electron transport by the phenolase complex. J. Am. Chem. Soc. 77,2914-15. Mauk, M.R., Barker, P.D., and Mauk A . G . (1991) Proton linkage of complex formation between cytochrome c and cytochrome bs'. electrostatic consequences of protein-protein interactions. Biochemistry 30, 9873-81. Mauk, M.R., Ferrer, J. and Mauk, A . G . (1994) Proton linkage in formation of the cytochrome c-cytochrome c peroxidase complex: Electrostatic properties of the high- and low-affinity cytochrome binding sites on the peroxidase. Biochemistry 33,12609-14. Mauk, A .G. , Mauk, M.R, Moore, G.R. and Northup, S .H. (1995) Experimental and theoretical analysis of the interaction between cytochrome c and cytochrome bs. J Bioenerg Biomembr 27, 311-30. McKee, T. C , Cardellina, J. H . , 2nd, Riccio, R., DAuria, M . V . , Iorizzi, M . , Minale, L. , Moran, R. A., Gulakowski, R. J., McMahon, J. B., Buckheit, R. W., Jr. and et al. (1994). HrV-inhibitory natural products. Comparative studies of sulfated sterols from marine invertebrates. J Med Chem 37, 793-7. 129 McRee, D. E. (1999). XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. J Struct Biol 125, 156-65. Mendel, S., Arndt, A. and Bugg, T. D. (2004). Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism: site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB). Biochemistry A3, 13390-6. Miyazaki K. and takenouchi M . (2002) Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. Biotechniques 33, 1033-4. Moore, G.R., Cox, M.C. , Crowe, D., Osborne, M.J., Rosell, F.I., Bujons, J., Barker, P.D., Mauk, M.R. and Mauk, A .G. (1998). N-epsilon,N-epsilon-dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein-protein complex formation. Biochem J 332, 439-49. Morris, G. M . , Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E. , Belew, R. K. and Olson, A. J. (1998). Automated Docking Using a Lamarckian Genetic Algorithm and Empirical Binding Free Energy Function. J. Comput. Chem. 19, 1639-1662. Mossa, J. S., El-Feraly, F. S. and Muhammad, I. (2004). Antimycobacterial constituents from Juniperus procera, Ferula communis and Plumbago zeylanica and their in vitro synergistic activity with isonicotinic acid hydrazide. Phytother Res 18, 934-7. Mulakala, C , Nerinckx, W. and Reilly, P. J. (2006). Docking studies on glycoside hydrolase Family 47 endoplasmic reticulum a-(l~>2)-mannosidase I to elucidate the pathway to the substrate transition state. Carbohydr Res 341, 2233-45. Muller, A. J., DuHadaway, J. B., Donover, P. S., Sutanto-Ward, E. and Prendergast, G. C. (2005a). Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Binl, potentiates cancer chemotherapy. Nat Med 11, 312-9. Muller, A. J., Malachowski, W; P. and Prendergast, G. C. (2005b). Indoleamine 2,3-dioxygenase in cancer: targeting pathological immune tolerance with small-molecule inhibitors. Expert Opin Ther Targets 9, 831-49. Munn, D. H., Shafizadeh, E. , Attwood, J. T., Bondarev, I., Pashine, A. and Mellor, A. L. (1999). Inhibition of T cell proliferation by macrophage tryptophan catabolism. J Exp Med 189, 1363-72. Munn, D. H. , Sharma, M . D., Hou, D., Baban, B., Lee, J. R., Antonia, S. J., Messina, J. L., Chandler, P., Koni, P. A. and Mellor, A. L. (2004). Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes. J Clin Invest 114,280-90. 130 Munn, D. H. , Sharma, M . D., Lee, J. R., Jhaver, K. G., Johnson, T. S., Keskin, D. B., Marshall, B., Chandler, P., Antonia, S. J., Burgess, R., Slingluff, C. L. , Jr. and Mellor, A. L. (2002). Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 297, 1867-70. Munn, D. FL, Zhou, M . , Attwood, J. T., Bondarev, I., Conway, S. J., Marshall, B., Brown, C. and Mellor, A. L. (1998). Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 281, 1191-3. Nakagawa, M . , Watanabe, H., Kodata, S., Okajima, H., Hino, T., Flippen, J. L. and Witkop, B. (1977) A valid model for the mechanism of oxidation of tryptophan to formylkynurenine-25 years later. Proc. Natl. Acad. Sci. U.S.A., 74,4730-33. Nakamura, M . , Kakuda, T., Oba, Y. , Ojika, M . and Nakamura, H. (2003). Synthesis of biotinylated xestoquinone that retains inhibitory activity against C a 2 + ATPase of skeletal muscle myosin. Bioorg Med Chem 11, 3077-82. Newman, D. J., Cragg, G. M . and Snader, K. M . (2000). The influence of natural products upon drug delivery. Nat. Prod. Rep. 17,215-234. Ni., F. (1994) Accounting for ligand-protein interactions in the relaxation-matrix analysis of transferred nuclear Overhauser effects. Prog. Nucl. Magn. Reson. Spectrosc. 26,517-606. Northrup, S.H., Thomasson, K.A. , Miller, C. M . , Barker, P.D., Eltis, L.D., Guillermette, J.G., Inglis, S.C. and Mauk A. G. (1993) Effects of charged amino acid mutations on the bimolecular kinetics of reduction of yeast iso-1-ferricytochrome c by bovine ferrocytochrome bs. Biochemistry 32, 6613-23. Myint, A. M . and Kim, Y. K. (2003). Cytokine-serotonin interaction through IDO: a neurodegeneration hypothesis of depression. Med Hypotheses 61, 519-25. Oda, S. I., Sugimoto, H., Yoshida, T. and Shiro, Y. (2006). Crystallization and preliminary crystallographic studies of human indoleamine 2,3-dioxygenase. Acta. Crystallograph Sect F Struct Biol Cryst Commun 62, 221-3. Oka, T. and Simpson, F. J. (1971). Quercetinase, a dioxygenase containing copper. Biochem Biophys Res Commun 43, 1-5. Okamoto, A., Nikaido, T., Ochiai, K., Takakura, S., Saito, M . , Aoki, Y. , Ishii, N., Yanaihara, N., Yamada, K., Takikawa, O., Kawaguchi, R., Isonishi, S., Tanaka, T. and Urashirha, M . (2005). Indoleamine 2,3-dioxygenase serves as a marker of poor prognosis in gene expression profiles of serous ovarian cancer cells. Clin Cancer Res 11, 6030-9. 131 Oliw, E. H. and Su, C. (1997). Catalytic and spectroscopic properties of linoleate diol synthase of the fungus Gaumannomyces graminis. Adv Exp Med Biol 433, 65-8. Omata, Y. , Sakamoto, H. , Robinson, R. C , Pincus, M . R. and Friedman, F. K. (1994). Interaction between cytochrome P450 2B1 and cytochrome bs- inhibition by synthetic peptides indicates a role for P450 residues Lys-122 and Arg-125. Biochem Biophys Res Commun 201, 1090-5. Oshino, N., Imai, Y. and Sato, R. (1971). A function of cytochrome bs in fatty acid desaturation by rat liver microsomes. J Biochem (Tokyo) 69, 155-67. Ozaki, Y. , Nichol, C. A. and Duch, D. S. (1987). Utilization of dihydroflavin mononucleotide and superoxide anion for the decyclization of L-tryptophan by murine epididymal indoleamine 2,3-dioxygenase. Arch Biochem Biophys 257, 207-16. Ozaki, Y. , Reinhard, J. F., Jr. and Nichol, C. A. (1986). Cofactor activity of dihydroflavin mononucleotide and tetrahydrobiopterin for murine epididymal indoleamine 2,3-dioxygenase. Biochem Biophys Res Commun 137, 1106-11. Paltauf, F., Esfandi, F. and Holasek, A. (1974). Stereospecificity of lipases. Enzymic hydrolysis of enantiomeric alkyl diacylglycerols by lipoprotein lipase, lingual lipase and pancreatic lipase. FEBS Lett 40, 119-23. Papadopoulou, N. D., Mewies, M . , McLean, K. J., Seward, H. E. , Svistunenko, D. A., Munro, A. W. and Raven, E. L. (2005). Redox and spectroscopic properties of human indoleamine 2,3-dioxygenase and a His303Ala variant: implications for catalysis. Biochemistry 44, 14318-28. Patil, S.D., Frelyer A.J., Breen A., Carte, B. and Johnson R.K. (1996). Halistanol disulfate B, novel sulfate sterol from the sponge Pachastrella sp.: inhibitor of endothelin converting enzyme. J Nat Prod 59, 606-8. Pereira, A., Vottero E. , Roberge, M . , Mauk, G.M. and Andersen, R. (2006) Indoleamine 2,3-dioxygenase inhibitors from the Northeastern Pacific Marine Hydroid Garveia annulata. J Nat Prod 69, 1496-9. Perkins, E. , Sun, D., Nguyen, A., Tulac, S., Francesco, M . , Tavana, H. , Nguyen, H., Tugendreich, S., Barthmaier, P., Couto, J., Yeh, E. , Thode, S., Jarnagin, K., Jain, A., Morgans, D. and Melese, T. (2001). Novel inhibitors of poly(ADP-ribose) polymerase/PARPl and PARP2 identified using a cell-based screen in yeast. Cancer Res 61,4175-83. Potula, R., Poluektova, L . , Knipe, B., Chrastil, J., Heilman, D., Dou, H. , Takikawa, O., Munn, D. H., Gendelman, H. E. and Persidsky, Y. (2005). Inhibition of 132 indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis. Blood 106, 2382-90. Poulos, T. L. and Mauk, A. G. (1983). Models for the complexes formed between cytochrome b$ and the subunits of methemoglobin. JBiol Chem 258, 7369-73. Reddy, V. V. , Kupfer, D. and Caspi, E. (1977). Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes. J Biol Chem 252, 2797'-801. Rizzuto, R., Brini, M . , De Giorgi, F., Rossi, R., Heim, R., Tsien, R.Y. and Pozzan, T. (1996) Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo. Curr Biol. 6,183-8. Rogers, J. P., Beuscher, A. E. t., Flajolet, M . , McAvoy, T., Nairn, A. C , Olson, A. J. and Greengard, P. (2006). Discovery of protein phosphatase 2C inhibitors by virtual screening. J Med Chem 49, 1658-67. Sakamoto, H., Furukawa, K., Matsunaga, K., Nakamura, H. and Ohizumi, Y. (1995). Xestoquinone activates skeletal muscle actomyosin ATPase by modification of the specific sulfhydryl group in the myosin head probably distinct from sulfhydryl groups SHI and SH2. Biochemistry 34, 12570-5. Salemme, F.R. (1976) An hypothetical structure for an intermolecular electron transfer complex of cytochromes c and bs. J. Mol. Biol 102, 563-8. Salter, M . , Hazelwood, R., Pogson, C. I., Iyer, R. and Madge, D. J. (1995). The effects of a novel and selective inhibitor of tryptophan 2,3-dioxygenase on tryptophan and serotonin metabolism in the rat. Biochem Pharmacol 49, 1435-42. Sardar, A. M . and Reynolds, G. P. (1995). Frontal cortex indoleamine-2,3-dioxygenase activity is increased in HIV-1-associated dementia. Neurosci Lett 187,9-12. Schenkman, J. B. and Jansson, I. (2003). The many roles of cytochrome bs- Pharmacol Ther 97, 139-52. Schuttelkopf, A. W. and van Aalten, D. M . (2004). PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr 60, 1355-63. Segel I.H. (1976) Biochemical Calculations. John Wiley & Sons, Inc. New York. pp. 257-61 Shet, M . S., Faulkner, K. M . , Holmans, P. L. , Fisher, C. W. and Estabrook, R. W. (1995). The effects of cytochrome bs, NADPH-P450 reductase, and lipid on the rate of 6 133 P-hydroxylation of testosterone as catalyzed by a human P450 3A4 fusion protein. Arch Biochem Biophys 318, 314-21. Shao, W., Im, S.C., Zuiderweg, E.R. and Waskell, L. (2003) Mapping the binding interface of the cytochrome 65-cytochrome c complex by nuclear magnetic resonance. Biochemistry 42, 14774-84. Shimakata, T., Mihara, K. and Sato, R. (1972). Reconstitution of hepatic microsomal stearoyl-coenzyme A desaturase system from solubilized components. J Biochem (Tokyo) 72, 1163-74. Shinzawa, K., Kominami, S. and Takemori, S. (1985). Studies on cytochrome P-450 (P-450 17 a,1 lyase) from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome bs. Biochim Biophys Acta 833, 151-60. Shteinman, A. A. (1995). The mechanism of methane and dioxygen activation in the catalytic cycle of methane monooxygenase. FEBS Lett 362, 5-9. Schenkman, J. B. and Jansson, I. (2003). The many roles of cytochrome bs. Pharmacol Ther 97, 139-52. Shu, L., Chiou, Y. M . , Orville, A. M . , Miller, M . A., Lipscomb, J. D. and Que, L. , Jr. (1995). X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism. Biochemistry 34, 6649-59. Simon, J. A. and Bedalov, A. (2004). Yeast as a model system for anticancer drug discovery. Nat Rev Cancer 4, 481 -92. So, S.-P., Wu, J., Huang, G., Huang, A., L i , D. and Ruan, K. -H. (2003). Identification of residues important for ligand binding of thromboxane A2 receptor in the second extracellular loop using the NMR experiment-guided mutagenesis approach. J Biol Chem 278, 10922-27. Solis, F. J. and Wets, J. B. (1981). Minimization by random search techniques Math. Oper Res 6, 19-30. Sono, M . (1989). Enzyme kinetic and spectroscopic studies of inhibitor and effector interactions with indoleamine 2,3-dioxygenase. 2. Evidence for the existence of another binding site in the enzyme for indole derivative effectors. Biochemistry 28,5400-7. Sono, M . and Cady, S. G. (1989). Enzyme kinetic and spectroscopic studies of inhibitor and effector interactions with indoleamine 2,3-dioxygenase. 1. Norharman and 4-phenylimidazole binding to the enzyme as inhibitors and heme ligands. Biochemistry 28, 5392-9. 134 Sono, M . and Dawson, J. H. (1984). Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy. Biochim Biophys Acta 7 8 9 , 170-87. Sono, M . , Roach, M . P., Coulter, E. D. and Dawson, J. H. (1996). Heme-containing oxygenases. Chem Rev 96,2841-2888. Sono, M . , Taniguchi, T., Watanabe, Y. and Hayaishi, O. (1980). Indoleamine 2,3-dioxygenase. Equilibrium studies of the tryptophan binding to the ferric, ferrous, and CO-bound enzymes. JBiol Chem 2 5 5 , 1339-45. Srinivas, P., Gopinath, G., Banerji, A., Dinakar, A. and Srinivas, G. (2004). Plumbagin induces reactive oxygen species, which mediate apoptosis in human cervical cancer cells. Mol Carcinog 4 0 , 201-11. Strittmatter, P., Spatz, L. , Corcoran, D., Rogers, M . J., Setlow, B. and Redline, R. (1974). Purification and properties of rat liver microsomal stearyl coenzyme A desaturase. Proc Natl Acad Sci US All, 4565-9. Su, C , Sahlin, M . and Oliw, E. H. (1998). A protein radical and ferryl intermediates are generated by linoleate diol synthase, a ferric hemeprotein with dioxygenase and hydroperoxide isomerase activities. JBiol Chem 2 7 3 , 20744-51. Sugie, S., Okamoto, K., Rahman, K. M , Tanaka, T., Kawai, K., Yamahara, J. and Mori, H. (1998). Inhibitory effects of plumbagin and juglone on azoxymethane-induced intestinal carcinogenesis in rats. Cancer Lett 1 2 7 , 177-83. Sugimoto, H., Oda, S. I., Otsuki, T., Hino, T., Yoshida, T. and Shiro, Y . (2006). Crystal structure of human indoleamine 2,3-dioxygenase: Catalytic mechanism of O2 incorporation by a heme-containing dioxygenase. Proc Natl Acad Sci USA. 103, 2611-6. Sugiyama, T., Miki, N., Miyake, Y. and Yamano, T. (1982). Interaction and electron transfer between cytochrome bs and cytochrome P-450 in the reconstituted p-nitroanisole O-demethylase system. J Biochem (Tokyo) 92, 1793-803. Sugiyama, T., Miki, N. and Yamano, T. (1980). N A D H - and NADPH-dependent reconstituted p-nitroanisole O-demethylation system containing cytochrome P-450 with high affinity for cytochrome bs. J Biochem (Tokyo) 8 7 , 1457-67. Sun, C , Aspland, S. E. , Ballatore, C , Castillo, R., Smith, A. B., 3rd and Castellino, A. J. (2006). The design, synthesis, and evaluation of two universal doxorubicin-linkers: preparation of conjugates that retain topoisomerase II activity. Bioorg Med Chem Lett 1 6 , 104-7. 135 Suzuki, T. (1994). Abalone myoglobins evolved from indoleamine dioxygenase: the cDNA-derived amino acid sequence of myoglobin from Nordotis madaka. J Protein Chem 13,9-13. Suzuki, T., Kawamichi, H. and Imai, K. (1998). A myoglobin evolved from indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 121,117-128. Suzuki, T. and Furukohri, T. (1989). The ear-shell (Sulculus diversicolor aquatilis) myoglobin is composed of an unusual 39 kDA polypeptide chain. Experientia 45, 998-1002. Suzuki, T., Kawamichi, H. and Imai, K. (1998). Amino acid sequence, spectral, oxygen-binding, and autoxidation properties of indoleamine dioxygenase-like myoglobin from the gastropod mollusc Turbo cornutus. J Protein Chem 17, 817-26. Takikawa, O., Kuroiwa, T., Yamazaki, F. and Kido, R. (1988). Mechanism of interferon-gamma action. Characterization of indoleamine 2,3-dioxygenase in cultured human cells induced by interferon-gamma and evaluation of the enzyme-mediated tryptophan degradation in its anticellular activity. J Biol Chem 263, 2041-8. Takikawa, O., Littlejohn, T., Jamie, J. F., Walker, M . J. and Truscott, R. J. (1999). Regulation of indoleamine 2,3-dioxygenase, the first enzyme in U V filter biosynthesis in the human lens. Relevance for senile nuclear cataract. Adv Exp Med Biol 467, 241-5. Takikawa, O., Littlejohn, T. K. and Truscott, R. J. (2001). Indoleamine 2,3-dioxygenase in the human lens, the first enzyme in the synthesis of U V filters. Exp Eye Res 72, 271-7. Takikawa, O., Truscott, R. J., Fukao, M . and Miwa, S. (2003). Age-related nuclear cataract and indoleamine 2,3-dioxygenase-initiated tryptophan metabolism in the human lens. A dv Exp Med Biol 527,277-85. Taniguchi, T., Sono, M . , Hirata, F., Hayaishi, O., Tamura, M . , Hayashi, K., Iizuka, T. and Ishimura, Y. (1979). Indoleamine 2,3-dioxygenase. Kinetic studies on the binding of superoxide anion and molecular oxygen to enzyme. JBiol Chem 254,3288-94. Terentis, A. C , Thomas, S. R., Takikawa, O., Littlejohn, T. K., Truscott, R. J., Armstrong, R. S., Yeh, S. R. and Stocker, R. (2002). The heme environment of recombinant human indoleamine 2,3-dioxygenase. Structural properties and substrate-ligand interactions. JBiol Chem 211, 15788-94. Terness, P., Bauer, T. M . , Rose, L. , Dufter, C , Watzlik, A., Simon, H. and Opelz, G. (2002). Inhibition of allogeneic T cell proliferation by indoleamine 2,3-136 dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J Exp Med 196,447-57. Tetef, M . , Margolin, K., Ann, C , Akman, S., Chow, W., Leong, L. , Morgan, R. J., Jr., Raschko, J., Somlo, G. and Doroshow, J. H. (1995). Mitomycin C and menadione for the treatment of lung cancer: a phase El trial. Invest New Drugs 13, 157-62. Tikkanen, L. , Matsushima, T., Natori, S. and Yoshihira, K. (1983). Mutagenicity of natural naphthoquinones and benzoquinones in the Salmonella/microsome test. Mutat Res 124, 25-34. True, R. G. and Lowe, J. E. (1980). Induced juglone toxicosis in ponies and horses. Am J Vet Res 41,944-5. Tsai, A. L. , Kulmacz, R. J., Wang, J. S., Wang, Y. , Van Wart, H. E. and Palmer, G. (1993). Heme coordination of prostaglandin H synthase. J Biol Chem 268, 8554-63. Tsang, P. K. S. and Donald, T. S. (1990). Electron-transfer thermodynamics and bonding for the superoxide (O2"), dioxygen (O2), and hydroxy (OH) adducts of (tetrakis (2,6-dichlorophenyl) porphinato) iron, -manganese, and -cobalt in dimethylformamide. Inorg. Chem. 29, 2848-2855. Tugendreich, S., Perkins, E. , Couto, J., Barthmaier, P., Sun, D., Tang, S., Tulac, S., Nguyen, A., Yeh, E . , Mays, A., Wallace, E. , Lila, T., Shivak, D., Prichard, M . , Andrejka, L. , Kim, R. and Melese, T. (2001). A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays. Genome Res 11, 1899-912. Uchiyama, T., Kanazawa, N. , Sasaki, T., Satoh, S., Abe, T. Takeuchi, T., Sadakata, H. , and Kobayashi, N. (1992) Inhibition of intravascular thrombin generation by warfarinn administrtion, with special reference to plasma thrombin-antithrombin rn complex (TAT) levels. Nippon Kessen Shiketsu Gakkaishi 3, 231-7. Ueno, M . , Kakinuma, Y. , Yuhki, K. I., Murakoshi, N., Iemitsu, M . , Miyauchi, T. and Yamaguchi, I. (2006). Doxorubicin induces apoptosis by activation of caspase-3 in cultured cardiomyocytes in vitro and rat cardiac ventricles in vivo. J Pharmacol Sci 101, 151-8. Uyttenhove, C , Pilotte, L. , Theate, I., Stroobant, V. , Colau, D., Parmentier, N., Boon, T. and Van den Eynde, B. J. (2003). Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 9, 1269-74. 137 Varga, Z., Bene, L., Pieri, C , Damjanovich, S. and Gaspar, R., Jr. (1996). The effect of juglone on the membrane potential and whole-cell K + currents of human lymphocytes. Biochem Biophys Res Commun 218, 828-32. Vatsis, K. P., Theoharides, A. D., Kupfer, D. and Coon, M . J. (1982). Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450. Participation of cytochrome b5. JBiol Chem 257, 11221-9. Verrax, J., Cadrobbi, J., Marques, C , Taper, H. , Habraken, Y. , Piette, J. and Calderon, P. B. (2004). Ascorbate potentiates the cytotoxicity of menadione leading to an oxidative stress that kills cancer cells by a non-apoptotic caspase-3 independent form of cell death. Apoptosis 9,223-33. Voet D., Voet J.D. and Pratt C.W. (1998) Fundaments of Biochemistry. John Wiley & Sons, Inc., USA, pp 340. Vottero, E. , Balgi A, Woods, K., Tugendreich, S. Melese, T., Andersen, R.J., Mauk, A . G . and Roberge, M . (2006) Inhibitors of human indoleamine 2,3-dioxygenase identified with a target based screen in yeast. Biotechnol J \ , 282-8. Vottero, E. Mitchell, D.A., Page, M.J., MacGillivray, R.T., Sadowski, I.J., Roberge, M . and Mauk A. G. (2006) Cytochrome b$ is a major reductant in vivo of human indoleamine 2,5-dioxygenase expressed in yeast. FEBS Lett. 580, 2265-8. Watanabe, Y., Fujiwara, M . , Yoshida, R. and Hayaishi, O. (1980). Stereospecificity of hepatic L-tryptophan 2,3-dioxygenase. Biochem J189, 393-405. Westley, A. M . and Westley, J. (1996). Enzyme inhibition in open systems. Superiority of uncompetitive agents. JBiol Chem 111, 5347-52. Wichers, M . C. and Maes, M . (2004). The role of indoleamine 2,3-dioxygenase (IDO) in the pathophysiology of interferon-a-induced depression. J Psychiatry Neurosci 29,11-7. Wykle, R. L. , Blank, M . L. , Malone, B. and Snyder, F. (1972). Evidence for a mixed function oxidase in the biosynthesis of ethanolamine plasmalogens from 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine. JBiol Chem 247, 5442-7 Yamamoto, S. and Hayaishi, O. (1967). Tryptophan pyrrolase of rabbit intestine. D- and L-tryptophan-cleaving enzyme or enzymes. JBiol Chem 242, 5260-6. 138 

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