Indoleamine 2,3-dioxygenase 2 (IDO2) and the kynurenine pathway: characteristics and potential roles in health and disease

The kynurenine pathway is the major route for the oxidative degradation of the amino acid tryptophan. Activity of the pathway is involved in several disease conditions, both in the periphery and the central nervous system, including cancer, inflammatory disorders, neurological conditions, psychiatric disorders and neurodegenerative diseases. Three enzymes are now known to catalyze the first and rate-limiting step in the catabolism of tryptophan along this pathway: tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO, subsequently named IDO1), both of which have been extensively studied, and a third enzyme, indoleamine 2,3-dioxygenase 2 (IDO2), a relative newcomer to the kynurenine pathway field. The adjuvant chemotherapeutic agent, 1-methyl-d-tryptophan, was intially suggested to target IDO2, implying involvement of IDO2 in tumorigenesis. Subsequently this compound has been suggested to have alternative actions and the physiological and pathophysiological roles of IDO2 are unclear. Targeted genetic interventions and selective inhibitors provide approaches for investigating the biology of IDO2. This review focuses on the current knowledge of IDO2 biology and discusses tools that will assist in further characterizing the enzymes of the kynurenine pathway.     

Changes in kynurenine pathway metabolism in the brain, liver and kidney of aged female Wistar rats

The kynurenine pathway of tryptophan catabolism plays an important role in several biological systems affected by aging. We quantified tryptophan and its metabolites kynurenine (KYN), kynurenine acid (KYNA), picolinic acid (PIC) and quinolinic acid (QUIN), and activity of the kynurenine pathway enzymes indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO) and quinolinic acid phosphoribosyltransferase (QPRTase), in the brain, liver and kidney of young, middle-aged and old female Wistar rats. Tryptophan levels and TDO activity decreased in all tissues with age. In contrast, brain IDO activity increased with age, while liver and kidney IDO activity decreased with age. The levels of KYN, KYNA, QUIN and PIC in brain all increased with age, while the levels of KYN in the liver and kidney showed a tendency to decrease. The levels of KYNA in the liver did not change, but the levels of KYNA in the kidney increased. The levels of PIC and QUIN increased significantly in the liver but showed a tendency to decrease in the kidney. QPRTase activity in both brain and liver decreased with age but was elevated in the kidney in middle-aged (12-month-old) rats. These age-associated changes in tryptophan metabolism have the potential to impact upon major biological processes, including lymphocyte function, pyridine (NAD(P)(H)) synthesis and N-methyl-d-aspartate (NMDA)-mediated synaptic transmission, and may therefore contribute to several degenerative changes of the elderly.     

Enzyme activities along the tryptophan-nicotinic acid pathway in alloxan diabetic rabbits

Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase.TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls.Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits.The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits.These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid-->nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.     

The kynurenine pathway modulates neurodegeneration in a Drosophila model of Huntington's disease

Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been implicated in the pathophysiology of neurodegenerative disorders, including Huntington's disease (HD) [1]. A central hallmark of HD is neurodegeneration caused by a polyglutamine expansion in the huntingtin (htt) protein [2]. Here we exploit a transgenic Drosophila melanogaster model of HD to interrogate the therapeutic potential of KP manipulation. We observe that genetic and pharmacological inhibition of kynurenine 3-monooxygenase (KMO) increases levels of the neuroprotective metabolite kynurenic acid (KYNA) relative to the neurotoxic metabolite 3-hydroxykynurenine (3-HK) and ameliorates neurodegeneration. We also find that genetic inhibition of tryptophan 2,3-dioxygenase (TDO), the first and rate-limiting step in the pathway, leads to a similar neuroprotective shift toward KYNA synthesis. Importantly, we demonstrate that the feeding of KYNA and 3-HK to HD model flies directly modulates neurodegeneration, underscoring the causative nature of these metabolites. This study provides the first genetic evidence that inhibition of KMO and TDO activity protects against neurodegenerative disease in an animal model, indicating that strategies targeted?at?two key points within the KP may have therapeutic relevance in HD, and possibly other neurodegenerative disorders.     

Synthesis of novel tryptanthrin derivatives as dual inhibitors of indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are promising drug development targets due to their implications in pathologies such as cancer and neurodegenerative diseases. The search for IDO1 inhibitor has been intensely pursued but there is a paucity of potent TDO and IDO1/TDO dual inhibitors. Natural product tryptanthrin has been confirmed to bear IDO1 and/or TDO inhibitory activities. Herein, twelve novel tryptanthrin derivatives were synthesized and evaluated for the IDO1 and TDO inhibitory potency. All of the compounds were found to be IDO1/TDO dual inhibitors, in particular, compound 9a and 9b bore IDO1 inhibitory activity similar to that of INCB024360, and compound 5a and 9b had remarkable TDO inhibitory activity superior to that of the well-known TDO inhibitor LM10. This work enriches the collection of IDO1/TDO dual inhibitors and provides chemical molecules for potential development into drugs.     

Substrate stereo‐specificity in tryptophan dioxygenase and indoleamine 2,3‐dioxygenase

The first and rate‐limiting step of the kynurenine pathway, in which tryptophan (Trp) is converted to N‐formylkynurenine is catalyzed by two heme‐containing proteins, Indoleamine 2,3‐dioxygenase (IDO), and Tryptophan 2,3‐dioxygenase (TDO). In mammals, TDO is found exclusively in liver tissue, IDO is found ubiquitously in all tissues. IDO has become increasingly popular in pharmaceutical research as it was found to be involved in many physiological situations, including immune escape of cancer. More importantly, small‐molecule inhibitors of IDO are currently utilized in cancer therapy. One of the main concerns for the design of human IDO (hIDO) inhibitors is that they should be selective enough to avoid inhibition of TDO. In this work, we have used a combination of classical molecular dynamics (MD) and hybrid quantum‐classical (QM/MM) methodologies to establish the structural basis that determine the differences in (a) the interactions of TDO and IDO with small ligands (CO/O₂) and (b) the substrate stereo‐specificity in hIDO and TDO. Our results indicate that the differences in small ligand bound structures of IDO and TDO arise from slight differences in the structure of the bound substrate complex. The results also show that substrate stereo‐specificity of TDO is achieved by the perfect fit of L ‐Trp, but not D ‐Trp, which exhibits weaker interactions with the protein matrix. For hIDO, the presence of multiple stable binding conformations for L /D ‐Trp reveal the existence of a large and dynamic active site. Taken together, our data allow determination of key interactions useful for the future design of more potent hIDO‐selective inhibitors. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

Comparative effects of oxygen on indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase of the kynurenine pathway

Indoleamine 2,3-dioxygenase (IDO) reacts with either oxygen or superoxide and tryptophan (trp) or other indoleamines while tryptophan 2,3-dioxygenase (TDO) reacts with oxygen and is specific for trp. These enzymes catalyze the rate-limiting step in the kynurenine (KYN) pathway from trp to quinolinic acid (QA) with TDO in kidney and liver and IDO in many tissues, including brain where it is low but inducible. QA, which does not cross the blood-brain barrier, is an excitotoxin found in the CNS during various pathologies and is associated with convulsions. We proposed that HBO-induced convulsions result from increased flux through the KYN pathway via oxygen stimulation of IDO. To test this, TDO and IDO of liver and brain, respectively, of Sprague Dawley rats were assayed with oxygen from 0 to 6.2 atm HBO. TDO activity was appreciable at even 30 microM oxygen and rose steeply to a maximum at 40 microM. Conversely, IDO had almost no detectable activity at or below 100 microM oxygen and maximum activity was not reached until about 1150 microM. (Plasma contains about 215 microM oxygen and capillaries about 20 microM oxygen when rats breathe air.) KYN was 60% higher in brains of HBO-convulsed rats compared to rats breathing air. While the oxygen concentration inside cells of rats breathing air or HBO is not known precisely, it is clear that the rate-limiting, IDO-catalyzed step in the brain KYN pathway (but not liver TDO) can be greatly accelerated in rats breathing HBO.     

Contributions of tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase to the conversion of d-tryptophan to nicotinamide analyzed by using tryptophan 2,3-dioxygenase-knockout mice

We investigated the contribution percentage of tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) to the conversion of d-tryptophan to nicotinamide in TDO-knockout mice. The calculated percentage conversions indicated that TDO and IDO oxidized 70 and 30%, respectively, of the dietary l-tryptophan. These results indicate that both TDO and IDO biosynthesize nicotinamide from d-tryptophan and l-tryptophan in mice.     

Reliable chromatographic assay for measuring of indoleamine 2,3-dioxygenase 1 (IDO1) activity in human cancer cells

The kynurenine pathway is the major tryptophan degradation routes generating bioactive compounds important in physiology and diseases. Depending on cell type it is initiated enzymatically by tryptophan-2,3-dioxygenase (TDO) or indoleamine-2,3-dioxygenase 1 and 2 (IDO1 and IDO2) to yield N-formylkynurenine as the precursor of further metabolites. Herein, we describe an accurate high-pressure liquid chromatography coupled with a diode array detector (HPLC-DAD) method to serve for IDO1 activity determination in human cancer cells cultured in vitro. Enzymatic activity was expressed as the rate of ʟ-kynurenine generation by 1 mg of proteins obtained from cancer cells. Our approach shows the limit of detection and limit of quantification at 12.9 and 43.0 nM Kyn, respectively. Applicability of this method was demonstrated in different cells (ovarian and breast cancer)exposed to various conditions and has successfully passed the validation process. This approach presents a useful model to study the role of kynurenine pathway in cancer biology.     

Evolution of Vertebrate Indoleamine 2,3-Dioxygenases

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the same reaction, the first step in tryptophan catabolism via the kynurenine pathway. TDO is widely distributed among life-forms, being found not only in eukaryotes but also in bacteria. In contrast, IDO has been found only in mammals and yeast to date. However, recent genome and EST projects have identified IDO homologues in non-mammals and found an IDO paralogue that is expressed in mice. In this study, we cloned the frog and fish IDO homologues and the mouse IDO paralogue, and characterized their enzymatic properties using recombinants. The IDOs of lower vertebrates and the mouse IDO paralogue had IDO activity but had 500–1000 times higher K _m values and very low enzyme efficiency compared with mammalian IDOs. It appears that L-Trp is not a true substrate for these enzymes in vivo, although their actual function is unknown. On the phylogenetic tree, these low-activity IDOs, which we have named “proto-IDOs,” formed a cluster that was distinct from the mammalian IDO cluster. The IDO and proto-IDO genes are present tandemly on the chromosomes of mammals, including the marsupial opossum, whereas only the proto-IDO gene is observed in chicken and fish genomes. These results suggest that (mammalian) IDOs arose from proto-IDOs by gene duplication that occurred before the divergence of marsupial and eutherian (placental) mammals in mammalian evolutionary history.     

Is tryptophan catabolism increased under indoleamine 2,3 dioxygenase activity during chronic lung inflammation in pigs?

In a preliminary study we observed that piglets suffering from chronic lung inflammation induced by an intravenous injection of complete Freund adjuvant showed a marked decrease in plasma tryptophan (Trp) concentration suggesting increased Trp utilisation. During the inflammatory process, a cytokine-induced enzyme called indoleamine 2,3-dioxygenase (IDO) has been shown to catabolise Trp into kynurenine (Kyn). Yet, during inflammation, increased Trp catabolism may decrease Trp availability for other functions such as growth. This metabolic pathway has never been studied in pigs. So, the objectives of this study were to measure IDO activity in pigs and to determine if the decrease in plasma Trp concentrations previously observed in piglets suffering from chronic lung inflammation could be explained by the induction of IDO activity. In order to do so, we compared IDO activity measured in the tracheo-bronchial lymph nodes and in the lungs of 7 piglets, injected with complete Freund adjuvant (CFA), to 7 pair-fed littermate healthy controls. Blood samples were taken at 0, 2, 5, 7 and 10 days following CFA injection in order to measure plasma Trp, Kyn and haptoglobin concentrations. Indoleamine 2,3-dioygenase activity in the tracheo-bronchial lymph nodes (P < 0.05), in the lungs (P < 0.07) and plasma haptoglobin (P < 0.01) were higher in pigs with lung inflammation than in the controls. Plasma Trp and Kyn were not significantly affected by CFA injection. Our data showed that IDO is activated under chronic lung inflammation in pigs. The impact of IDO activation on plasma Trp concentration and its availability is discussed according to the amount of Trp provided by the diet.     

Identification of selective inhibitors of indoleamine 2,3-dioxygenase 2

The kynurenine pathway is responsible for the breakdown of the majority of the essential amino acid, tryptophan (Trp). The first and rate-limiting step of the kynurenine pathway can be independently catalysed by tryptophan 2,3-dioxygenase (Tdo2), indoleamine 2,3-dioxygenase 1 (Ido1) or indoleamine 2,3-dioxygenase 2 (Ido2). Tdo2 or Ido1 enzymatic activity has been implicated in a number of actions of the kynurenine pathway, including immune evasion by tumors. IDO2 is expressed in several human pancreatic cancer cell lines, suggesting it also may play a role in tumorigenesis. Although Ido2 was originally suggested to be a target of the chemotherapeutic agent dextro-1-methyl-tryptophan, subsequent studies suggest this compound does not inhibit Ido2 activity. The development of selective Ido2 inhibitors could provide valuable tools for investigating its activity in tumor development and normal physiology. In this study, a library of Food and Drug Administration-approved drugs was screened for inhibition of mouse Ido2 enzymatic activity. A number of candidates were identified and IC₅₀ values of each compound for Ido1 and Ido2 were estimated. The Ido2 inhibitors were also tested for inhibition of Tdo2 activity. Our results showed that compounds from a class of drugs used to inhibit proton pumps were the most potent and selective Ido2 inhibitors identified in the library screen. These included tenatoprazole, which exhibited an IC₅₀ value of 1.8 μM for Ido2 with no inhibition of Ido1 or Tdo2 activity detected at a concentration of 100 μM tenatoprazole. These highly-selective Ido2 inhibitors will be useful for defining the distinct biological roles of the three Trp-catabolizing enzymes.     

Drosophila eye color mutants as therapeutic tools for Huntington disease

Huntington disease (HD) is a fatal inherited neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (htt). A pathological hallmark of the disease is the loss of a specific population of striatal neurons, and considerable attention has been paid to the role of the kynurenine pathway (KP) of tryptophan (TRP) degradation in this process. The KP contains three neuroactive metabolites: 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and kynurenic acid (KYNA). 3-HK and QUIN are neurotoxic, and are increased in the brains of early stage HD patients, as well as in yeast and mouse models of HD. Conversely, KYNA is neuroprotective and has been shown to be decreased in HD patient brains. We recently used a Drosophila model of HD to measure the neuroprotective effect of genetic and pharmacological inhibition of kynurenine monoxygenase (KMO)—the enzyme catalyzing the formation of 3-HK at a pivotal branch point in the KP. We found that KMO inhibition in Drosophila robustly attenuated neurodegeneration, and that this neuroprotection was correlated with reduced levels of 3-HK relative to KYNA. Importantly, we showed that KP metabolites are causative in this process, as 3-HK and KYNA feeding experiments modulated neurodegeneration. We also found that genetic inhibition of the upstream KP enzyme tryptophan-2,3-dioxygenase (TDO) was neuroprotective in flies. Here, we extend these results by reporting that genetic impairment of KMO or TDO is protective against the eclosion defect in HD model fruit flies. Our results provide further support for the possibility of therapeutic KP interventions in HD.     

Drosophila eye color mutants as therapeutic tools for Huntington disease

Huntington disease (HD) is a fatal inherited neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (htt). A pathological hallmark of the disease is the loss of a specific population of striatal neurons, and considerable attention has been paid to the role of the kynurenine pathway (KP) of tryptophan (TRP) degradation in this process. The KP contains three neuroactive metabolites: 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and kynurenic acid (KYNA). 3-HK and QUIN are neurotoxic, and are increased in the brains of early stage HD patients, as well as in yeast and mouse models of HD. Conversely, KYNA is neuroprotective and has been shown to be decreased in HD patient brains. We recently used a Drosophila model of HD to measure the neuroprotective effect of genetic and pharmacological inhibition of kynurenine monoxygenase (KMO)-the enzyme catalyzing the formation of 3-HK at a pivotal branch point in the KP. We found that KMO inhibition in Drosophila robustly attenuated neurodegeneration, and that this neuroprotection was correlated with reduced levels of 3-HK relative to KYNA. Importantly, we showed that KP metabolites are causative in this process, as 3-HK and KYNA feeding experiments modulated neurodegeneration. We also found that genetic inhibition of the upstream KP enzyme tryptophan-2,3-dioxygenase (TDO) was neuroprotective in flies. Here, we extend these results by reporting that genetic impairment of KMO or TDO is protective against the eclosion defect in HD model fruit flies. Our results provide further support for the possibility of therapeutic KP interventions in HD.     

Induction of tryptophan 2,3-dioxygenase in the mouse endometrium during implantation

Indoleamine 2,3-dioxygenase (IDO) is expressed in trophoblasts and defends the conceptus against rejection by reducing the tryptophan level and suppressing the T cell activity. We isolated a cDNA for tryptophan 2,3-dioxygenase (TDO), another key catabolizing enzyme of tryptophan, from a mouse uterus cDNA library enriched with pregnancy-induced genes. Northern blot and in situ hybridization analyses demonstrated that the TDO mRNA was induced in the decidualized stromal cells around the implanted embryo at the time of implantation. The expression was then upregulated and primarily localized at the mesometrial decidua. TDO mRNA was induced by deciduoma formation as well as embryo transfer but not by ovarian steroid hormones. These findings demonstrated that TDO is induced in the endometrial stromal cells concomitant with decidualization and suggested its involvement in the implantation process by regulating the tryptophan level at the implantation site.     

Design,synthesis and structure-activity relationship study of novel naphthoindolizine and indolizinoquinoline-5,12-dione derivatives as IDO1 inhibitors

Indoleamine 2,3-dioxygenase 1 (IDO1) is regarded as a promising target for cancer immunotherapy. Many naphthoquinone derivatives have been reported as IDO1 inhibitors so far. Herein, two series of naphthoquinone derivatives, naphthoindolizine and indolizinoquinoline-5,12-dione derivatives, were synthesized and evaluated for their IDO1 inhibitory activity. Most of the target compounds showed significant inhibition potency and high selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO). The structure-activity relationship was also summarized. The most potent compounds 5c (IC₅₀ 23?nM, IDO1 enzyme), and 5b′ (IC₅₀ 372?nM, HeLa cell) were identified as promising lead compounds.     

Multiple roles of haem in cystathionine β-synthase activity: implications for hemin and other therapies of acute hepatic porphyria

The role of haem in the activity of cystathionine β-synthase (CBS) is reviewed and a hypothesis postulating multiple effects of haem on enzyme activity under conditions of haem excess or deficiency is proposed, with implications for some therapies of acute hepatic porphyrias. CBS utilises both haem and pyridoxal 5′-phosphate (PLP) as cofactors. Although haem does not participate directly in the catalytic process, it is vital for PLP binding to the enzyme and potentially also for CBS stability. Haem deficiency can therefore undermine CBS activity by impairing PLP binding and facilitating CBS degradation. Excess haem can also impair CBS activity by inhibiting it via CO resulting from haem induction of haem oxygenase 1 (HO 1), and by induction of a functional vitamin B₆ deficiency following activation of hepatic tryptophan 2,3-dioxygenase (TDO) and subsequent utilisation of PLP by enhanced kynurenine aminotransferase (KAT) and kynureninase (Kynase) activities. CBS inhibition results in accumulation of the cardiovascular risk factor homocysteine (Hcy) and evidence is emerging for plasma Hcy elevation in patients with acute hepatic porphyrias. Decreased CBS activity may also induce a proinflammatory state, inhibit expression of haem oxygenase and activate the extrahepatic kynurenine pathway (KP) thereby further contributing to the Hcy elevation. The hypothesis predicts likely changes in CBS activity and plasma Hcy levels in untreated hepatic porphyria patients and in those receiving hemin or certain gene-based therapies. In the present review, these aspects are discussed, means of testing the hypothesis in preclinical experimental settings and porphyric patients are suggested and potential nutritional and other therapies are proposed.     

Distinct Tryptophan Catabolism and Th17/Treg Balance in HIV Progressors and Elite Controllers

Tryptophan (Trp) catabolism into immunosuppressive kynurenine (Kyn) by indoleamine 2,3-dioxygenase (IDO) was previously linked to Th17/Treg differentiation and immune activation. Here we examined Trp catabolism and its impact on Th17/Treg balance in uninfected healthy subjects (HS) and a large cohort of HIV-infected patients with different clinical outcomes: ART-naïve, Successfully Treated (ST), and elite controllers (EC). In ART-naïve patients, increased IDO activity/expression, together with elevated levels of TNF-α and sCD40L, were associated with Treg expansion and an altered Th17/Treg balance. These alterations were normalized under ART. In contrast, Trp 2,3-dioxegenase (TDO) expression was dramatically lower in EC when compared to all other groups. Interestingly, EC displayed a distinctive Trp metabolism characterized by low Trp plasma levels similar to ART-naïve patients without accumulating immunosuppressive Kyn levels which was accompanied by a preserved Th17/Treg balance. These results suggest a distinctive Trp catabolism and Th17/Treg balance in HIV progressors and EC. Thus, IDO-induced immune-metabolism may be considered as a new inflammation-related marker for HIV-1 disease progression.