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Tryptophan 2,3-dioxygenase (TDO), along with indoleamine 2,3-dioxygenase (IDO) and indoleamine 2,3-dioxygenase-2 (IDO2) are the three enzymes that catalyse oxidation of L-tryptophan (L-Trp) in the first step of the kynurenine pathway. Despite the fact that all three catalyse the same reaction, they were detected and characterized in different chronological periods; TDO, IDO and IDO2 were discovered in 1936, 1967 and 2007 respectively. Years of studies showed that abnormal regulation of L-Trp, in the first step of kynurenine pathway, is related with several disorders, including cancer. Regardless of their distinct dissimilarities, TDO, IDO and IDO2 were all detected in various cancers, supporting tumour escape and survival. The early identification of IDO immunomodulatory action (1990s) led to intense research for the development of IDO inhibitors, but not TDO. Despite this effort, the most pharmacologically suitable IDO inhibitor, 1-methyltryptophan (1- MT), appears to be ineffective as monotherapeutic drug. Discovery of IDO2 showed that 1-MT action is not fully understood, raising questions about the biological significance of IDO2. The ultimate goal of the current study was to address the problems outlined above. Because TDO and IDO are two druggable molecular targets, the discovery of a new class of effective inhibitors was pursued. Plate screening of ~2800 potential inhibitor compounds obtained from National Cancer Institute (NCI), USA, indicated seven promising compounds that inhibit both TDO and IDO in either nanomolar or low micromolar range. Interestingly, of these seven inhibitors, six have been identified to have cytotoxic action against several types of tumour cell lines (NCI data). NSC 26326, known as b-lapachone, is a natural occurring quinone and the strongest inhibitor of all seven. This NCI compound inhibits both TDO and IDO with inhibition constants of ~30-70 nM and 97 ± 14 nM respectively. Like NSC 26326, NSC 36398 is another natural occurring product and the only compound that showed selectivity against TDO with inhibition constant of 16.3 ± 3.8 μM. Among the seven compounds that displayed promise as inhibitors of TDO and IDO was mitomycin C. Mitomycin C, which is an approved oncology drug and a known inhibitor of IDO (Ki = 24.2 ± 1.2 μM), is also inhibitor of TDO with inhibition constant of 2.86 ± 0.03 μM. Another major goal of the current work was the discovery of isatin derivatives as inhibitors of TDO and IDO. Using the tryptophan-like structure of isatin as starting point, a number of structural modifications were carried out (structureactivity relationship (SAR)) succeeding the optimization of their inhibition activity. This new family of TDO and IDO inhibitors demonstrated inhibition potencies in the low micromolar range with 5,7-dicholoisatin to reach the nanomolar range (in the case of TDO). Halogenation of isatin and its derivatives was found to increase noticeably the inhibition potencies of these molecules by 12fold and 6fold for TDO and IDO respectively while breakdown of isatin’s pyrrolidine ring had a disastrous result on the inhibition of both enzymes. Combinations of 1-MT with either the newly-identified NCI inhibitors or the isatin derivatives were also examined. The in vitro combinations of 1-MT with either the NCI inhibitors or the isatin derivatives revealed an additive effect without though excluding the possibility of synergistic effect in vivo. The specificity of TDO, IDO and IDO2 against the two stereoisomers of 1- MT was also investigated, with interesting results. While IDO is inhibited only by the L-isoform of 1-MT (Ki = 18.0 ± 3.4 μM), IDO2 is inhibited by both 1-Me-L-Trp and 1-Me-D-Trp with inhibition constants of 306 ± 17 μM and 3419 ± 259 μM respectively. Biochemical characterization of human IDO2 was another goal of the current thesis, which completed successfully. Kinetic, redox and inhibition study of human IDO2 indicated significant differences in comparison with human IDO something which suggests the potential implication of IDO2 in an identified biological pathway (other than tryptophan catabolism function).The findings presented herein help to solve the mystery of 1-MT action, at least in vitro, give answers in regards to IDO2 function, and provide a number of new, promising inhibitors for TDO and IDO.

Indoleamine 2 3-dioxygenase (IDO) has recently been highlighted as a promising target for small molecule based immunotherapy. IDO is often coopted by various cancer cells to promote an immune-suppressive environment around tumors. DNA damage repair (DDR) enzymes have recently been targeted for inhibition to promote genetic instability and bolster immune recognition. DDR enzymes such as PARP and POLγ are common inhibition targets due to their direct effects on cellular function. In the process of designing conjugate inhibitors of IDO and DDR enzymes, novel synthetic methodology was developed for the mild deprotection of N-Tert-butyloxycarbonyl (N-BOC) group from various amines. Conjugate inhibitors of IDO and PARP were synthesized using the parent molecules 1-MLT and Olaparib. The conjugate molecules showed more potent cytotoxicity in various cancer cell lines than their precursor molecules. The novel conjugate inhibitors could be used as probes to explore interplay between the PARP and IDO signaling pathways and elucidate any synergy or dependency between the two enzymes.

Cancer is a critical public health issue in the United States and immunotherapy has emerged as a breakthrough in providing innovative care to patients. Many cancers evade immune detection by altering the ratio of serum L-tryptophan (L-Trp) to L-kynurenine (L-Kyn), an intermediate in tryptophan catabolism that inhibits antitumoral immunity. Earlier, the Georgiou lab demonstrated that administration of P. fluorescens kynureninase (PƒKYNU) results in systematic L-Kyn depletion, thereby relieving immune suppression and inhibiting in vivo tumor progression. However, PƒKYNU has low amino acid identity to the native human kynureninase enzyme, and is likely to be immunogenic, and therefore unsuitable for human therapy. Unfortunately, the human kynureninase (HsKYNU) has poor catalytic activity for the hydrolysis of L-kynurenine. In the first part of this dissertation, I describe my engineering efforts of the Mucilaginibacter paludis kynureninase (MpKYNU), which has higher homology to the human enzyme. In parallel, I performed in vitro studies to gain insight into how L-Kyn affects T cells and specifically how it creates immunosuppression through the induction of non-classical regulatory T cells, thereby contributing to a critical gap of knowledge in the field while advancing our understanding of KYNU’s therapeutic efficacy.

Drug development is an ever-increasingly prominent field in the scientific community. A crucial step in the drug development process involves examining the toxicity of potential therapeutic compounds. A plethora of organic compounds have been synthesized through an ongoing drug discovery program at Ohio University. Two of these compounds have demonstrated significant promise in several models of disease. In the present study, the potential toxicity of these two compounds was evaluated via in silico and in vitro techniques.

Indoleamine 2,3-dioxygenase (IDO) is an important immunoregulatory heme enzyme that controls T-cell responses during diverse disease conditions by catalyzing tryptophan degradation along the kynurenine pathway. IDO is expressed within inflammatory or hypoxic tissues, characterized by oxidative stress or low oxygen tension, respectively. These studies investigated how reduction and oxidation reactions control IDO under oxidative or hypoxic conditions. The oxidant hydrogen peroxide (H2O2) potently inhibited dioxygenase activity by activating IDO’s peroxidase function, leading to compound I-mediated protein oxidation, an opening of the distal active-site pocket and non-productive substrate binding, changes inhibited by tryptophan. Protection by tryptophan involved its compound I-dependent oxidation into oxindolylalanine and kynurenine and binding to IDO compound II, retarding peroxidase turnover. Physiological peroxidase substrates ascorbate and nitrite also inhibited dioxygenase inactivation, despite the latter promoting IDO tyrosine nitration. Pathological oxidants, peroxynitrite and hypochlorite, inhibited IDO dioxygenase activity with reduced efficacy compared to H2O2, reflecting their propensity to directly oxidize IDO protein and inability to form compound I. Stopped flow spectroscopy revealed H2O2 and peroxynitrite react with ferric-IDO with rate-constants of 8.1x10^3 and 5.1x10^4 M^-1 s^-1, respectively. The reaction of peroxidase substrates with IDO compounds I and II revealed unique kinetic profiles for each substrate, with an overall order of reactivity: tyrosine >ascorbate >urate >nitrite. Ascorbate accelerated IDO compound II formation upon reaction of ferric-IDO with H2O2 but not peroxynitrite, indicating compound I formation is specific for H2O2. Under hypoxia, IDO efficiently reduced nitrite to nitric oxide (NO) in a nitrite-, enzyme concentration- and pHdependent manner via its nitrite reductase activity (rate-constant: 5.1 M^-1 s^-1). EPR spectroscopy and NO-spin trapping showed that the ascorbate and methylene blue reducing co-factors supported IDO’s nitrite reductase activity and NO release that was inhibited by tryptophan, 1-methyl-L-tryptophan, but not 1-methyl-D-tryptophan. In vitro experiments demonstrated that nitrite supplementation of IDO-expressing endothelial cells reduced IDO protein levels and dioxygenase activity under hypoxic, but not normoxic, conditions. These biochemical studies identify IDO as a multi-functional heme enzyme able to catalyze peroxidase and nitrite reductase reactions. Moreover, IDO is sensitive to redox regulation by physiological oxidants and peroxidase substrates, which have important implications for IDO’s biological actions.

The mammalian gut bacterial microbiome is increasingly recognized as a vital immune organ. Numerous studies in murine models implicate the gut bacterial microbiome not only as being required for the induction and maintenance of various important cellular mediators of immunity, but also as being capable of initiating and/or exacerbating states of chronic inflammation in the gut and the periphery. HIV infection and subsequent disease progression is driven by chronic inflammation, and is concomitantly characterized by a disruption within many mucosal cellular compartments involved in regulating gut microbial community composition, including Paneth cells, macrophages, dendritic cells, and Th17 cells. Previous work from our laboratory has shown a link between Th17 cell depletion and activity of the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO1), which produces molecules known as kynurenines that can directly inhibit differentiation of Th17 cells in vitro and in vivo. However, a putative interaction between the gut microbiome and the constellation of immune disruptions in HIV, including chronic inflammation and mucosal immune disruption (perhaps involving the IDO1 pathway), had not yet been described. We hypothesized that HIV infection is associated with a shift in gut microbiome composition, and that this shift could potentiate further mucosal immune disruptions and the chronic inflammation that drives HIV disease progression. We profiled the gut mucosal-adherent microbiome of 32 human subjects at varying stages of HIV disease. We found that the microbiomes of untreated, infected individuals was significantly different from those that were uninfected, and the extent to which this microbiome differed was proportionally related to markers of chronic inflammation and mucosal immune disruption (IDO1 activity). Furthermore, we found that some gut bacterial taxa that were enriched in HIV infection correlated strongly and positively with IDO1 activity, and representative members of the top such correlate could directly produce kynurenines in vitro. We further sought to identify gut-resident bacteria that may inhibit IDO1 activity, and longitudinally profiled 12 rhesus macaques before and after SIV infection toward that end. We found that fecal abundance of Lactobacillus was negatively correlated with IDO1 activity, and macaques given a Lactobacillus-containing probiotic exhibited diminished IDO1 activity as measured in peripheral blood.

Abstract: Infants that develop severe bronchiolitis due to Respiratory Syncytial Virus (RSV) are at increased risk of developing asthma later in life. To begin my studies in vitro, purified RSV stock was needed. My first study established the critical steps in RSV purification to determine a procedure that ensures the removal of potential contaminating pro-inflammatory mediators in viral preparations. Using polyethylene glycol and ultracentrifugation through various sucrose gradient concentrations, we collected samples at all steps of purification to determine the RSV titer, total protein (µg/mL) and pro-inflammatory cytokines (ELISA). We analyzed the efficacy of each step and determined that, regardless of optimal purification methods employed, CCL5, a bioactive chemokine in allergic inflammation, persisted in virus preparations and co-purified with RSV. This conclusion is important for research on RSV or allergic diseases. In the second part of the study, the basis of the association between RSV and the development of allergic inflammation was investigated. The tryptophan catabolizing enzyme indoleamine 2, 3-dioxygenase (IDO) induces apoptosis of Th1, but not Th2 cells, which may contribute to immune responses associated with allergy and asthma. I hypothesized that RSV induces IDO in human dendritic cells, which results in a Th2-biased immune profile. Human peripheral blood monocytes from healthy adult donors were isolated, differentiated to dendritic cells (moDC), in vitro, and infected with RSV. RSV induced IDO activity and this effect was inhibited by Palivizumab, UV-inactivation and Ribavarin. Inhibition of endosomal TLR function with chloroquine did not block IDO activity. The signal transduction cascade for RSV-induced IDO activity was initiated by intracellular pattern recognition receptors (i.e., RIG-I related) via NF-κB and p38 MAPK pathways. In a transwell system, co-culture of RSV infected moDC with activated T-cells suppressed t-bet (a Th1-associated factor) but not GATA3 (Th2 regulator) expression. Inhibition of IDO activity with the competitive inhibitor, 1-methyl tryptophan, blocked this effect on t-bet expression. In conclusion, this study showed that RSV induced the expression and bioactivity of IDO in moDC, in a virus replication-dependant fashion. My study suggests that RSV has the capacity to play a role in increasing Th2-type response via IDO.

Hematopoietic stem cell transplant is the only curative therapy for a variety of malignant and non-malignant hematologic disorders and has the potential to treat a variety of other difficult diseases. Unfortunately, the procedure carries a high risk of graft versus host disease (GVHD), limiting its efficacy and wider application. GVHD occurs when antigen disparity between donor and host leads donor T cells to become activated and proliferate. They then migrate to the liver, skin, lung, and gut and mediate tissue damage. By uncovering the positive and negative regulatory pathways in GVHD biology, new therapeutic targets can be identified. Data presented here establishes indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme in the tryptophan metabolism pathway, as a major regulator of GVHD. IDO-deficient recipients of bone marrow transplant suffered accelerated GVHD lethality, with pathology concentrated in the colon. IFN-γ produced by donor T cells highly upregulated colon IDO; in its absence, donor T cells underwent more proliferation and less apoptosis, leading to increased disease severity and lethality. Tryptophan depletion was not central to these effects, as T cells lacking the sensor for tryptophan were suppressed by IDO. Accordingly, treatment of transplant recipients with kynurenines, the downstream metabolites of tryptophan breakdown, ameliorated disease. IDO expressed by antigen presenting cells (APCs) was responsible for its T cell suppressive effects. By targeting APCs using a TLR agonist, IDO could be upregulated in the colon before transplantation, and this upregulation was sufficient to diminish GVHD. This work identifies IDO as a potent modulator of GVHD. This finding is then used to develop two clinically relevant therapeutic strategies for GVHD treatment and prevention.