Kynurenine 3-monooxygenase is a FAD-dependent aromatic hydroxylase (FAH) which is a widely suggested therapeutic target for controlling the balance of bioactive metabolite levels produced by the mammalian kynurenine pathway (KP). Prior to starting this work no structural information was known for the enzyme, with studies of the human form complicated by the presence of a C-terminal transmembrane helix. The bacterial Pseudomonas fluorescens enzyme (PfKMO) lacks the transmembrane region and has been previously characterised by Crozier-Reabe and Moran [1, 2]. Therefore PfKMO, which shares 32 % sequence identity with the human enzyme, was selected as a target for structure solution. Initial substrate bound PfKMO crystals showed poor X-ray diffraction. Subsequent growth optimisation and the generation of a C252S/C461S PfKMO mutant (dm2) yielded crystals suitable for structure solution. Selenomethioninelabelled substrate bound dm2 crystals were used to solve the first structure to a resolution of 3.40 Å. With just one protein molecule per asymmetric unit, a high solvent content was responsible for the poor diffraction properties of this crystal form. The overall fold resembled that of other FAH enzymes with a Rossmann-fold based FADbinding domain above a buried substrate binding pocket. Interestingly PfKMO possesses an additional, novel C-terminal domain that caps the back of the substrate-binding pocket on the opposite side to the flavin. Residues proposed to be involved in substrate binding were identified and shown to be highly conserved among mammalian KMO sequences. Subsequently single crystals of substrate-free dm2 PfKMO were obtained and showed significantly stronger diffraction due to new lattice packing in an orthorhombic space group bearing four molecules per asymmetric unit. The structure was solved to a resolution of 2.26 Å and revealed a clear conformational change of the novel C-terminal domain. This movement opens a potential route of substrate/product exchange between bulk solvent and the active site. The investigation of a set of C-terminal mutants further explored the relevance and mechanics of the conformational change. In addition the presence of chloride ions in the substrate-free crystal growth solution caused a small number of localised subtle alterations to the structure, with a potential chloride binding site identified adjacent to the flavin cofactor. This may have relevance for the observed inhibition of PfKMO activity by monovalent anions – a feature widely common to FAH enzymes [3]. The first discovered KMO inhibitors were analogues of the substrate L-Kyn, however one such compound (m-NBA) was recently shown to instigate uncoupled NADPH oxidation leading to the release of cytotoxic hydrogen peroxide [1]. A set of substrate analogues were tested and characterised for inhibition of PfKMO. The picture was shown to be complex as some substrate analogues completely inhibited the enzyme whilst the binding of some still stimulated low-levels of NADPH oxidation. Crystallographic studies with m-NBA and 3,4-dichlorobenzoylalanine (3,4-CBA) bound revealed indistinguishable structures from that of substrate-bound PfKMO. These studies suggest that the analogue 3,4CBA is a potent PfKMO inhibitor whose therapeutic potential may be re-visited. The previous most potent KMO inhibitor whose structure was not analogous to the substrate was Ro 61-8048 [4], which unfortunately did not pass pre-clinical safety tests. A novel series of 1,2,4-oxadiazole amides based on the structure of Ro 61-8048 was created by Gavin Milne (PhD, University of St Andrews) and tested on PfKMO. Rounds of refinement led to the discovery and refinement of low nanomolar competitive inhibitors of the bacterial enzyme. PfKMO was co-crystallised with each of the four most potent compounds forming a third different lattice arrangement, which yielded structures to resolutions of 2.15-2.40 Å. The structures displayed conformational changes resembling the substrate-free fold possibly caused by displacement of a crucial substrate-binding residue, R84. Overall the wealth of structural data obtained may be transferable to predictions about the structural features of human KMO and to the rational design of therapeutic inhibitors. The potent novel inhibitors tested may additionally present a new exciting development for the therapeutic inhibition of human KMO.

Kynurenine 3-monooxygenase (KMO) is a flavoprotein which has been implicated in Huntington’s disease, Alzheimer’s disease and acute pancreatitis. Recently there has been important research published about this enzyme including the structure of a truncated Saccharomyces cerevisiae KMO enzyme and KMO inhibition studies in animal models of disease. In previous work from this research group the complete Pseudomonas fluorescens KMO enzyme has been successfully crystallised both with and without the substrate, L-kynurenine, from which significant insights were gained into function and the potential role of domain movement. To examine substrate binding in KMO and to consolidate previous structural studies, key residues in the active site have been investigated using site directed mutagenesis, crystallography and kinetic analysis using steady-state techniques. This analysis has identified the interactions between the enzyme and the substrate and provides a basis for inhibitor design. The residues implicated in substrate binding are N369, Y404 and R84. For N369 and Y404, minor changes to the amino acid in the mutations N369S and Y404F were shown to cause a decrease in binding affinity of the substrate but the enzyme remained active. For the mutations Y404A and R84K enzyme activity was significantly affected. Crystal structures of N369S, Y404F and R84K were also obtained. Another residue in the active site studied was H320 which is the only amino acid to differ in the active sites of the human and Pseudomonas fluorescens enzymes. This residue was therefore of interest to determine whether the bacterial enzyme used in this work is likely to be a good model for the human enzyme, which has not yet been successfully isolated in significant quantities for in vitro research. Modifying this residue to obtain H320F KMO revealed that this residue does not have a significant role in substrate binding. Potent inhibitor molecules have been studied with this enzyme and shown in kinetic assays to have nanomolar Ki values. These inhibitors are the most potent inhibitors studied with Pseudomonas fluorescens to date and continue previous inhibitor studies carried out with this enzyme. This group of inhibitors contain different substituents in the part of the molecule shown to bind closest to the C-terminal domain of the protein. These novel inhibitors do not allow the flavin to be reduced by NADPH (which results in unwanted peroxide production) unlike a number of previously studied molecules and therefore have the potential to be clinically useful. This research therefore answers many questions about this enzyme, in particular about the role of particular residues in the active site, substrate recognition and inhibition of this important drug target.

The study evaluated whether the activity of the indoleamine 2,3 dioxygenase (IDO) enzyme is increased post-stroke and contributes to the development of post-stroke depression (PSD) via tryptophan (TRP) depletion and neurotoxic kynurenine (KYN) metabolite production. The activity of IDO was measured using the KYN/TRP ratio. Participants were assessed for depression severity using the Center for Epidemiological Studies Depression Scale (CES-D). Blood TRP, KYN, large neutral amino acids and cytokines were measured and compared. Fifty-four (mean age=69.9±15.2, male=52.7%, mean NIHSS=7.3±4.6) patients within 28.9±40.3 days of stroke were separated into two groups: non-depressed (n=38, CES-D=6.1±4.9) and those with significant depressive symptoms (n=16, CES-D=26.8±10.8). Higher mean KYN/TRP ratios were demonstrated in stroke patients with depressive symptoms (non-depressed=69.3±36.9 vs. depressive symptoms=78.3±42.0, F3,50=4.61, p=0.006) after controlling for LNAA (p=0.026) and hypertension (p=0.039). As the KYN/TRP ratio reflects decreased TRP and increased neurotoxic KYN metabolites, both mechanisms may play an etiological role in PSD.

The study evaluated whether the activity of the indoleamine 2,3 dioxygenase (IDO) enzyme is increased post-stroke and contributes to the development of post-stroke depression (PSD) via tryptophan (TRP) depletion and neurotoxic kynurenine (KYN) metabolite production. The activity of IDO was measured using the KYN/TRP ratio. Participants were assessed for depression severity using the Center for Epidemiological Studies Depression Scale (CES-D). Blood TRP, KYN, large neutral amino acids and cytokines were measured and compared. Fifty-four (mean age=69.9±15.2, male=52.7%, mean NIHSS=7.3±4.6) patients within 28.9±40.3 days of stroke were separated into two groups: non-depressed (n=38, CES-D=6.1±4.9) and those with significant depressive symptoms (n=16, CES-D=26.8±10.8). Higher mean KYN/TRP ratios were demonstrated in stroke patients with depressive symptoms (non-depressed=69.3±36.9 vs. depressive symptoms=78.3±42.0, F3,50=4.61, p=0.006) after controlling for LNAA (p=0.026) and hypertension (p=0.039). As the KYN/TRP ratio reflects decreased TRP and increased neurotoxic KYN metabolites, both mechanisms may play an etiological role in PSD.

BACKGROUND: Acute pancreatitis (AP) is a sterile initiator of systemic inflammation that can trigger multiple organ dysfunction syndrome (MODS). In the acute phase of AP, the kynurenine pathway of tryptophan metabolism plays an important role in the genesis of AP-MODS in experimental animal models, but it is unknown whether the pathway is activated in human AP. Human data are required to support the rationale for kynurenine 3- monooxygenase (KMO) inhibition as a treatment for AP-MODS and reinforce the translational potential. Additionally, as respiratory dysfunction is frequent in severe AP, the role of lung ultrasonography in severity stratification deserves investigation. Furthermore, the effect of AP-MODS on long-term survival is unknown. OBJECTIVES: My objectives were to: 1) Define the temporal and quantitative relationship of kynurenine metabolites with the onset and severity of APMODS, 2) Investigate the value of lung ultrasonography in the early diagnosis of respiratory dysfunction in human AP-MODS, and 3) Examine whether early AP-MODS impacts on long-term survival. METHODS: 1) A prospective, observational, clinical experimental medicine study titled “Inflammation, Metabolism, and Organ Failure in Acute Pancreatitis” (IMOFAP) was performed. For 90 days, consecutive patients with a potential diagnosis of AP were recruited and venous blood was sampled at 0, 3, 6, 12, 24, 48, 72 and 168 hours post-recruitment. Kynurenine metabolite concentrations were measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and analysed in the context of clinical data, disease severity indices, and cytokine profiles. 2) In a nested cohort within IMOFAP, 41 participants underwent lung ultrasonography to evaluate whether this imaging modality can detect respiratory dysfunction in AP. 3) Survival data for a prospectively maintained database of patients with AP was analysed after accounting for in-hospital deaths. RESULTS: 1) During the IMOFAP study, 79 patients were recruited with an elevated serum amylase, of which 57 patients met the diagnostic criteria for AP; 9 had severe disease. Temporal profiling revealed early tryptophan depletion and contemporaneous elevation of plasma concentrations of 3- hydroxykynurenine, which paralleled systemic inflammation and AP severity. 2) Lung ultrasonography findings correlated with respiratory dysfunction. 3) 694 patients were followed up for a median of 8.8 years. AP-MODS conferred a deleterious effect on overall survival which persisted after the exclusion of inhospital deaths (10.0 years, 95% C.I. = 9.4-10.6 years) compared to AP without MODS (11.6 years, 95% C.I. = 11.2-11.9 years; P = 0.001). This effect was independent of age. CONCLUSIONS: In the acute phase of AP, metabolic flux through KMO is elevated and proportionate to AP severity. Lung ultrasonography may be a useful technique for evaluating AP-MODS. AP-MODS is an independent predictor of long-term mortality. Together, this work reinforces the rationale for investigating early phase KMO inhibition as a therapeutic strategy in humans.

Dans ce travail de thèse, nous nous sommes intéressés à mieux comprendre le rôle du métabolisme du tryptophane (TRP), et en particulier de la voie de la kynurénine (KYN), dans la physiopathologie des troubles dépressifs en utilisant un modèle murin de dépression - le stress chronique imprédictible modéré (Unpredictable Chronic Mild Stress = UCMS). Nous avons montré que 1) l'UCMS affecte de façon différentielle le métabolisme de la KYN selon qu'il se déroule en périphérie ou dans le SNC 2) l'UCMS induit l'accumulation de certains métabolites toxiques de la KYN en périphérie alors que dans le cerveau, l'effet est structure-dépendant 3) la concentration en KYN est inversement proportionnelle à la concentration en 5-HT dans le SNC 4) que l'activation de la voie KYN périphérique est positivement corrélée à l'expression de comportements anxio-dépressifs 5) que l'inhibition pharmacologique de la voie KYN a des effets antidépresseurs. / During this thesis, we were interested in better understand the role of the kynurenine pathway (KP) in the pathophysiology of depressive disorders by using a murine model of depression - the Unpredictable Chronic Mils StressProcedure = UCMS). We have shown that 1) UCMS has different effects on peripheral and cerebral tissues 2) UCMS induces accumulation of some toxic KP metabolites in the periphery and the CNS 3) the cerebral level of KYN innegatively correlated to the level of 5-HT 4) activation of the peripheral KP is positively correlated to the expression of anxiety-like and depressive-like behaviors, only in UCMS mice 5) pharmacological inhibition of the KP have antidepressant properties.

The kynurenine pathway plays a critical role in regulating immunological homeostasis in the brain. Evidence supporting the hypothesis that kynurenine pathway dysfunction may exacerbate progression of neurodegenerative diseases like Parkinson’s is growing. First, we investigate the effects of Interferon-γ, Lipopolysaccharide, and Interleukin-4 on several key kynurenine pathway metabolites using high performance liquid chromatography. We found that Interferon-γ had significant effects on the extracellular concentration of kynurenine metabolites in astrocytes, microglia, and macrophage. GCSF gene therapy is previously demonstrated to exert neuroprotective effects on models of Parkinson’s and Alzheimer’s disease. Seven days after receiving GCSF gene therapy, A53T Parkinson’s mice were found to have increased levels of GCSF and tyrosine hydroxylase positive neurons. A concurrent increase in expression of the kynurenine pathway enzyme kynurenine aminotransferase 2 was observed. GCSF gene therapy may exhibit neuroprotective effects in a Parkinson’s disease mouse model by restoring this key kynurenine pathway enzyme. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Kynurenine (KYN) is the first stable metabolite of the kynurenine pathway, which accounts for over 95% of tryptophan metabolism. Two previous studies by this research group reported elevated plasma KYN in Tourette syndrome (TS) patients when compared with age and sex matched controls and another study showed that KYN potentiated 5-HT2A-mediated head-shakes (HS) in rodents. These movements have been suggested to model tics in TS. This raised the questions how KYN acts in eliciting this response and whether it is an action of its own or of a further metabolite along the kynurenine pathway. In the liver, where most of the kynurenine pathway metabolism takes place under physiological conditions, the first and the rate limiting enzyme is tryptophan-dioxygenase (TDO) which can be induced by cortisol. In extrahepatic tissues the same step of the pathway is catalyzed by indoleamine-dioxygenase (IDO), which is induced by cytokines, predominantly interferon-y (INF-y). Plasma neopterin, which shows parallel increase with KYN following immune stimulation, was also found elevated in one of these studies positively correlating with KYN. In the present work animal studies suggested that KYN potentiates and quinolinic acid (QUINA) dose dependently inhibits the 5-HT2A-mediated HS response in mice. The potentiating effect seen with KYN was suggested to be an effect of KYN itself. Radioligand binding and phosphoinositide (PI) hydrolysis studies were done to explore the mechanisms by which kynurenine pathway metabolites could alter a 5-HT2A-receptor mediated response. None of the kynurenine pathway metabolites tested showed direct binding to 5-HT2A-receptors. PI hydrolysis studies with KYN and QUINA showed that KYN did not have any effect while QUINA inhibited 5-HT2A-mediated PI hydrolysis. Plasma cortisol determination in TS patients with elevated plasma KYN did not show elevated plasma cortisol levels, suggesting that the increase of plasma KYN in these TS patients is unlikely to be due to an increased TDO activity induced by increased cortisol. Attention deficit hyperactivity disorder (ADHD) is commonly associated with TS. Salivary cortisol detected in a group of children primarily affected with ADHD showed significantly lower salivary cortisol levels when compared with age and sex matched controls. Plasma tryptophan, KYN, neopterin, INF-y and KYN/tryptophan ratio and night-time urinary 6-sulphatoxymelatonin (aMT6s) excretion measured in a group of TS patients did not show any difference in their levels when compared with age and sex matched controls, but TS patients failed to show the expected positive correlation seen between plasma INF-y, neopterin and KYN and the negative correlation seen between plasma KYN and night-time urinary aMT6s excretion seen in healthy controls. The relevance of the kynurenine pathway, melatonin secretion and cortisol to Tourette Syndrome and associated conditions and the mechanism by which KYN and QUINA alter the 5-HT2A-receptor mediated HS response are discussed.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease of unclear aetiology, although the general consensus is of a multifactorial disease. The kynurenine pathway (KP), activated during neuroinflammation, is emerging as a possible contributory factor in ALS. The KP is the major route for tryptophan (TRP) catabolism. The intermediates generated can be either neurotoxic, such as quinolinic acid (QUIN), or neuroprotective, such as picolinic acid (PIC), an important endogenous metal chelator. The first and inducible enzyme is indoleamine 2,3-dioxygenase (IDO). As the extent of the involvement of the KP in ALS is unknown, the main aim of this thesis was to attempt to answer that question. The techniques used in this work include HPLC, GC/MS, RT-PCR, immunohistochemistry and immunocyctochemsitry. The main findings of this project are: (1) the complete KP is present in the mouse motor neuron cell line, NSC-34; (2) QUIN toxicity on NSC-34 cells may be ameliorated through the administration of NMDA antagonists, neuroprotective kynurenines, kynurenine inhibitor and QUIN monoclonal antibody; (3) in ALS patients, QUIN CSF and serum levels are significantly elevated, while PIC serum levels are significantly reduced; (4) ALS brain and spinal cord tissue show extensive microglia activation and positive immunoreactivity IDO and QUIN in spinal motor neurons and Betz cells in the motor cortex; and (5) kynurenine pathway inhibitor and analogue, R061-8048 and tranilast, are able to prolong the survival in the G93A SOD1 ALS transgenic mouse model. In conclusion, this study provide the first strong evidence for the involvement of the KP in ALS, and these data point to an inflammation-driven excitotoxic-chelation defective mechanism in ALS, which is amenable to KP analogue and inhibitor in ALS transgenic mice.

Kynurenine 3-monooxygenase (K3MO) lies on the kynurenine pathway, the major pathway for the catabolism of L-tryptophan. It converts kynurenine to 3-hydroxy kynurenine. Inhibition of K3MO is important in several neurological diseases and there is evidence that inhibition of K3MO could also be targeted for the prevention of multiple organ failure, secondary to acute pancreatitis. A structure activity relationship based upon the 1,2,4-oxadiazoles motif was carried out which revealed amide 207 as an inhibitor of P. fluorescens K3MO. Further structure activity relationships were developed based upon 207. This revealed 3,4-dichloro substitution in 235 and 245 as optimum for inhibition. Co-crystalisation of these inhibitors with P. fluorescens K3MO revealed their interactions with the enzyme. It also highlighted new, potential interactions between the inhibitors and K3MO. This led to the synthesis of 271 and 272, which were also potent inhibitors of K3MO. These amides were successfully co-crystalised with P. fluorescens K3MO. Further development of the amides followed, with amide 282 providing the most potent inhibitor of P. fluorescens K3MO to date (Kᵢ = 29.1 nM).