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Significance: TDO2 is more highly expressed than the nonhomologous TRP-catabolizing enzyme IDO1 in TNBC. We find that TDO2 knockdown can lead to a compensatory increase in IDO1. Therefore, we tested a newly developed TDO2/IDO1 dual inhibitor and found that it decreases TRP catabolism, anchorage-independent survival, and invasive capacity., (©2024 The Authors; Published by the American Association for Cancer Research.)

Background: This study aimed to identify the roles of L-tryptophan (Trp) and its rate-limiting enzymes on the receptivity of bovine endometrial epithelial cells. Real-time PCR was conducted to analyze the differential expression of genes between different groups of bovine endometrial epithelial cells. Western blot was performed to detect Cyclooxygenase-2 (COX2) expression after treatment with Trp or kynurenine (the main metabolites of Trp). The kynurenine assay was used to examine if Trp or prostaglandin E2 (PGE2) can increase the production of kynurenine in the bovine endometrial epithelial cells., Results: Trp significantly stimulates insulin growth factor binding protein 1 (IGFBP1) expression, a common endometrial marker of conceptus elongation and uterus receptivity for ruminants. When bovine endometrial epithelial cells are treated with Trp, tryptophan hydroxylase-1 remains unchanged, but tryptophan 2,3-dioxygenase 2 (TDO2) is significantly increased, suggesting tryptophan is mainly metabolized through the kynurenine pathway. Kynurenine significantly stimulates IGFBP1 expression. Furthermore, Trp and kynurenine significantly increase the expression of aryl hydrocarbon receptor (AHR). CH223191, an AHR inhibitor, abrogates the induction of Trp and kynurenine on IGFBP1. PGE2 significantly induces the expression of TDO2, AHR, and IGFBP1., Conclusions: The regulation between Trp / kynurenine and PGE2 may be crucial for the receptivity of the bovine uterus., (© 2024. The Author(s).)

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are attractive drug targets for cancer immunotherapy. After disappointing results of the epacadostat as a selective IDO inhibitor in phase III clinical trials, there is much interest in the development of the TDO selective inhibitors. In the current study, several data analysis methods and machine learning approaches including logistic regression, Random Forest, XGBoost and Support Vector Machines were used to model a data set of compounds retrieved from ChEMBL. Models based on the Morgan fingerprints revealed notable fragments for the selective inhibition of the IDO, TDO or both. Multiple fragment docking was performed to find the best set of bound fragments and their orientation in the space for efficient linking. Linking the fragments and optimization of the final molecules were accomplished by means of an artificial intelligence generative framework. Finally, selectivity of the optimized molecules was assessed and the top 4 lead molecules were filtered through PAINS, Brenk and NIH filters. Results indicated that phenyloxalamide, fluoroquinoline, and 3-bromo-4-fluroaniline confer selectivity towards the IDO inhibition. Correspondingly, 1-benzyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione was found to be an integral fragment for the selective inhibition of the TDO by constituting a coordination bond with the Fe atom of heme. In addition, furo[2,3-c]pyridine-2,3-diamine was found as a common fragment for inhibition of the both targets and can be used in the design of the dual target inhibitors of the IDO and TDO. The new fragments introduced here can be a useful building blocks for incorporation into the selective TDO or dual IDO/TDO inhibitors., Competing Interests: Declaration of competing interest All authors declare that they have no potential conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Human tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are two important targets in cancer immunotherapy. Extensive research has led to a large number of potent IDO inhibitors; in addition, 52 structures of IDO in complex with inhibitors with a wide array of chemical scaffolds have been documented. In contrast, progress in the development of TDO inhibitors has been limited. Only four structures of TDO in complex with competitive inhibitors that compete with the substrate L-tryptophan for binding to the active site have been reported to date. Here we systematically evaluated the structures of TDO in complex with competitive inhibitors with three types of pharmacophores, imidazo-isoindole, indole-tetrazole, and indole-benzotriazole. The comparative assessment of the protein-inhibitor interactions sheds new light into the structure-based design of enzyme-selective inhibitors.

Despite the immune system's role in the detection and eradication of abnormal cells, cancer cells often evade elimination by exploitation of various immune escape mechanisms. Among these mechanisms is the ability of cancer cells to upregulate amino acid-metabolizing enzymes, or to induce these enzymes in tumor-infiltrating immunosuppressive cells. Amino acids are fundamental cellular nutrients required for a variety of physiological processes, and their inadequacy can severely impact immune cell function. Amino acid-derived metabolites can additionally dampen the anti-tumor immune response by means of their immunosuppressive activities, whilst some can also promote tumor growth directly. Based on their evident role in tumor immune escape, the amino acid-metabolizing enzymes glutaminase 1 (GLS1), arginase 1 (ARG1), inducible nitric oxide synthase (iNOS), indoleamine 2,3-dioxygenase 1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and interleukin 4 induced 1 (IL4I1) each serve as a promising target for immunotherapeutic intervention. This review summarizes and discusses the involvement of these enzymes in cancer, their effect on the anti-tumor immune response and the recent progress made in the preclinical and clinical evaluation of inhibitors targeting these enzymes., Competing Interests: Author YG was employed by Oncolines B.V., (Copyright © 2024 Grobben.)

Tryptophan dioxygenase (TDO) and indoleamine 2,3 dioxygenase (IDO) belong to a unique class of heme-based enzymes that insert dioxygen into the essential amino acid, L-tryptophan (Trp), to generate N-formylkynurenine (NFK), a critical metabolite in the kynurenine pathway. Recently, the two dioxygenases were recognized as pivotal cancer immunotherapeutic drug targets, which triggered a great deal of drug discovery targeting them. The advancement of the field is however hampered by the poor understanding of the structural properties of the two enzymes and the mechanisms by which the structures dictate their functions. In this review, we summarize recent findings centered on the structure, function, and dynamics of the human isoforms of the two enzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Glioblastoma is an aggressive brain cancer, usually of unknown etiology, and with a very poor prognosis. Survival from diagnosis averages only 3 months if left untreated and this only increases to 12-15 months upon treatment. Treatment options are currently limited and typically comprise radiotherapy plus a course of the DNA-alkylating chemotherapeutic temozolomide. Unfortunately, the disease invariably relapses after several months of treatment with temozolomide, due to the development of resistance to the drug. Increased local tryptophan metabolism is a feature of many solid malignant tumours through increased expression of tryptophan metabolising enzymes. Glioblastomas are notable for featuring increased expression of the tryptophan catabolizing enzymes indole-2,3-dioxygenase-1 (IDO1), and especially tryptophan-2,3-dioxygenase-2 (TDO2). Increased IDO1 and TDO2 activity is known to suppress the cytotoxic T cell response to tumour cells, and this has led to the proposal that the IDO1 and TDO2 enzymes represent promising immuno-oncology targets. In addition to immune modulation, however, recent studies have also identified the activity of these enzymes is important in the development of resistance to chemotherapeutic agents., Methods: In the current study, the efficacy of a novel dual inhibitor of IDO1 and TDO2, AT-0174, was assessed in an orthotopic mouse model of glioblastoma. C57BL/6J mice were stereotaxically implanted with GL261(luc2) cells into the striatum and then administered either vehicle control, temozolomide (8 mg/kg IP; five 8-day cycles of treatment every 2 days), AT-0174 (120 mg/kg/day PO) or both temozolomide + AT-0174, all given from day 7 after implantation., Results: Temozolomide decreased tumour growth and improved median survival but increased the infiltration of CD4 + Tregs. AT-0174 had no significant effect on tumour growth or survival when given alone, but provided clear synergy in combination with temozolomide, further decreasing tumour growth and significantly improving survival, as well as elevating CD8 + T cell expression and decreasing CD4 + Treg infiltration., Conclusion: AT-0174 exhibited an ideal profile for adjunct treatment of glioblastomas with the first-line chemotherapeutic drug temozolomide to prevent development of CD4 + Treg-mediated chemoresistance., (© 2024. The Author(s).)

Glioblastoma (GBM) continues to exhibit a discouraging survival rate despite extensive research into new treatments. One factor contributing to its poor prognosis is the tumor's immunosuppressive microenvironment, in which the kynurenine pathway (KP) plays a significant role. This study aimed to explore how KP impacts the survival of newly diagnosed GBM patients. We examined tissue samples from 108 GBM patients to assess the expression levels of key KP markers-tryptophan 2,3-dioxygenase (TDO2), indoleamine 2,3-dioxygenase (IDO1/2), and the aryl hydrocarbon receptor (AhR). Using immunohistochemistry and QuPath software, three tumor cores were analyzed per patient to evaluate KP marker expression. Kaplan-Meier survival analysis and stepwise multivariate Cox regression were used to determine the effect of these markers on patient survival. Results showed that patients with high expression of TDO2, IDO1/2, and AhR had significantly shorter survival times. This finding held true even when controlling for other known prognostic variables, with a hazard ratio of 3.393 for IDO1, 2.775 for IDO2, 1.891 for TDO2, and 1.902 for AhR. We suggest that KP markers could serve as useful tools for patient stratification, potentially guiding future immunomodulating trials and personalized treatment approaches for GBM patients., (© 2024. The Author(s).)

The L-tryptophan (Trp) transport system is highly selective for Trp with affinity in the nanomolar range. This transport system is augmented in human interferon (IFN)-γ-treated and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells. Up-regulated cellular uptake of Trp causes a reduction in extracellular Trp and initiates immune suppression. Recent studies demonstrate that both IDO1 and tryptophanyl-tRNA synthetase (TrpRS), whose expression levels are up-regulated by IFN-γ, play a pivotal role in high-affinity Trp uptake into human cells. Furthermore, overexpression of tryptophan 2,3-dioxygenase (TDO2) elicits a similar effect as IDO1 on TrpRS-mediated high-affinity Trp uptake. In this review, we summarize recent findings regarding this Trp uptake system and put forward a possible molecular mechanism based on Trp deficiency induced by IDO1 or TDO2 and tryptophanyl-AMP production by TrpRS., (© 2024 The Author(s).)

Indoleamine 2,3-dioxygenase 1 (IDO) is a tryptophan (Trp) metabolic enzyme along the kynurenine (NFK) pathway. Under pathological conditions, IDO overexpressed by tumor cells causes depletion of tryptophan and the accumulation of metabolic products, which inhibit the local immune response and form immune escape. Therefore, the suppression of IDO activity is one of the strategies for tumor immunotherapy, and drug design for this target has been the focus of research for more than two decades. Apart from IDO, tryptophan dioxygenase (TDO) of the same family can also catalyze the same biochemical reaction in the human body, but it has different tissue distribution and substrate selectivity from IDO. Based on the principle of drug design with high potency and low cross-reactivity to specific targets, in this subject, the activity and selectivity of IDO and TDO toward small molecular inhibitors were studied from the perspective of thermodynamics and kinetics. The aim was to elucidate the structural requirements for achieving favorable biological activity and selectivity of IDO and TDO inhibitors. Specifically, the interactions of inhibitors from eight families with IDO and TDO were initially investigated through molecular docking and molecular dynamics simulations, and the thermodynamic data for binding of inhibitors were predicted by the molecular mechanics/generalized Born surface area (MM/GBSA) method. Secondly, we explored the free energy landscape of JKloops, the kinetic control element of IDO/TDO, using temperature replica exchange molecular dynamics (T-REMD) simulations and elucidated the connection between the rules of IDO/TDO conformational changes and the inhibitor selectivity mechanism. Furthermore, the binding and dissociation processes of the C1 inhibitor (NLG919) were simulated by the adaptive steering molecular dynamics (ASMD) method, which not only addressed the possible stable, metastable, and transition states for C1 inhibitor-IDO/TDO interactions, but also accurately predicted kinetic data for C1 inhibitor binding and dissociation. In conclusion, we have constructed a complete process from enzyme (IDO/TDO) conformational activation to inhibitor binding/dissociation and used the thermodynamic and kinetic data of each link as clues to verify the control mechanism of IDO/TDO on inhibitor selectivity. This is of great significance for us to understand the design principles of tumor immunotherapy drugs and to avoid drug resistance of immunotherapy drugs.

Neuroblastoma poses significant challenges in clinical management. Despite its relatively low incidence, this malignancy contributes disproportionately to cancer-related childhood mortality. Tailoring treatments based on risk stratification, including MYCN oncogene amplification, remains crucial, yet high-risk cases often confront therapeutic resistance and relapse. Here, we explore the aryl hydrocarbon receptor (AHR), a versatile transcription factor implicated in diverse physiological functions such as xenobiotic response, immune modulation, and cell growth. Despite its varying roles in malignancies, AHR's involvement in neuroblastoma remains elusive. Our study investigates the interplay between AHR and its ligand kynurenine (Kyn) in neuroblastoma cells. Kyn is generated from tryptophan (Trp) by the activity of the enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2). We found that neuroblastoma cells displayed sensitivity to the TDO2 inhibitor 680C91, exposing potential vulnerabilities. Furthermore, combining TDO2 inhibition with retinoic acid or irinotecan (two chemotherapeutic agents used to treat neuroblastoma patients) revealed synergistic effects in select cell lines. Importantly, clinical correlation analysis using patient data established a link between elevated expression of Kyn-AHR pathway genes and adverse prognosis, particularly in older children. These findings underscore the significance of the Kyn-AHR pathway in neuroblastoma progression, emphasizing its potential role as a therapeutic target., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Discovering effective anti-cancer agents poses a formidable challenge given the limited efficacy of current therapeutic modalities against various cancer types due to intrinsic resistance mechanisms. Cancer immunochemotherapy is an alternative strategy for breast cancer treatment and overcoming cancer resistance. Human Indoleamine 2,3-dioxygenase (hIDO1) and human Tryptophan 2,3-dioxygenase 2 (hTDO2) play pivotal roles in tryptophan metabolism, leading to the generation of kynurenine and other bioactive metabolites. This process facilitates the de novo synthesis of Nicotinamide Dinucleotide (NAD), promoting cancer resistance. This study identified a new dual hIDO1/hTDO2 inhibitor using a drug repurposing strategy of FDA-approved drugs. Herein, we delineate the development of a ligand-based pharmacophore model based on a training set of 12 compounds with reported hIDO1/hTDO2 inhibitory activity. We conducted a pharmacophore search followed by high-throughput virtual screening of 2568 FDA-approved drugs against both enzymes, resulting in ten hits, four of them with high potential of dual inhibitory activity. For further in silico and in vitro biological investigation, the anti-hypercholesterolemic drug Pitavastatin deemed the drug of choice in this study. Molecular dynamics (MD) simulations demonstrated that Pitavastatin forms stable complexes with both hIDO1 and hTDO2 receptors, providing a structural basis for its potential therapeutic efficacy. At nanomolar (nM) concentration, it exhibited remarkable in vitro enzyme inhibitory activity against both examined enzymes. Additionally, Pitavastatin demonstrated potent cytotoxic activity against BT-549, MCF-7, and HepG2 cell lines (IC 50  = 16.82, 9.52, and 1.84 µM, respectively). Its anticancer activity was primarily due to the induction of G1/S phase arrest as discovered through cell cycle analysis of HepG2 cancer cells. Ultimately, treating HepG2 cancer cells with Pitavastatin affected significant activation of caspase-3 accompanied by down-regulation of cellular apoptotic biomarkers such as IDO, TDO, STAT3, P21, P27, IL-6, and AhR., (© 2024. The Author(s).)

Tryptophan catabolism plays an important role in the metabolic reconnection in cancer cells to support special demands of tumor initiation and progression. The catabolic product of the tryptophan pathway, kynurenine, has the capability of suppressing the immune reactions of tumor cells. In this study, we conducted internal and external cohort studies to reveal the importance of tryptophan 2,3-dioxygenase (TDO) for lung adenocarcinoma (LUAD). Our study further demonstrated that the TDO2 expression was associated with the proliferation, survival, and invasion of LUAD cells, and targeting TDO2 for LUAD tumors could be a potential therapeutic strategy.

Objective: Fibroids are characterized by marked overexpression of tryptophan 2,3 dioxygenase (TDO2). The objective of this study was to determine the effectiveness of in vivo administration of an inhibitor of TDO2 (680C91) on fibroid size and gene expression., Design: Animal and ex vivo human study., Setting: Academic Research Institution., Subjects: Severe combined immunodeficiency mice bearing human fibroid xenografts treated with vehicle and TDO2 inhibitor., Intervention: Daily intraperitoneal administration of 680C91 or vehicle for 2 months and in vitro studies with fibroid explants., Main Outcome Measures: Tumor weight and gene expression profile of xenografts and in vitro mechanistic experiments using fibroid explants., Results: Compound 680C91 was well-tolerated with no effects on blood chemistry and body weight. Treatment of mice with 680C91 resulted in 30% reduction in the weight of fibroid xenografts after 2 months of treatment and as expected lower levels of kynurenine, the byproduct of tryptophan degradation and an endogenous ligand of aryl hydrocarbon receptor (AhR) in the xenografts. The expression of cytochrome P450 family 1 subfamily B member 1 (CYP1B1), transforming growth factor β3 (TGF-β3), fibronectin (FN1), cyclin-dependent kinase 2 (CDK2), E2F transcription factor 1 (E2F1), interleukin 8 (IL-8) and secreted protein acidic and cysteine rich (SPARC) mRNA were lower in the xenografts of mice treated with 680C91 compared with vehicle controls. Similarly, the protein abundance of collagen, FN1, CYP1B1, and SPARC were lower in the xenografts of 680C9- treated mice compared with vehicle controls. Immunohistochemical analysis of xenografts indicated decreased expression of collagen, Ki67 and E2F1 but no significant changes in cleaved caspase 3 expression in mice treated with 680C91. The levels of kynurenine in the xenografts showed a direct correlation with the tumor weight and FN1 levels. In vitro studies with fibroid explants showed a significant induction of CYP1B1, TGF-β3, FN1, CDK2, E2F1, IL8, and SPARC mRNA by tryptophan, which could be blocked by cotreatment with 680C91 and the AhR antagonist CH-223191., Conclusion: The results indicate that correction of aberrant tryptophan catabolism in fibroids could be an effective treatment through its effect to reduce cell proliferation and extracellular matrix accumulation., Competing Interests: Declaration of Interests T.D.C. and O.K. report a patent “Use of Tryptophan 2,3 dioxygenase (TDO2) inhibitors for treatment of fibroids” was applied in 2021. N.T. has nothing to disclose. S.R. has nothing to disclose. D.K. has nothing to disclose. D.B. has nothing to disclose., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Tolerogenic dendritic cells are promising for restoring immune homeostasis and may be an alternative therapy for autoimmune diseases such as rheumatoid arthritis. The kynurenine pathway is a vital mechanism that induces tolerance in dendritic cells (DCs). Tryptophan 2,3-dioxygenase (TDO2) is an important rate-limiting enzyme in the kynurenine pathway and participates in immune regulation. However, the role of TDO2 in shaping the tolerogenic phenotypes of DCs remains unclear. In this study, we investigated the effects and mechanisms of TDO2-overexpressed DCs in regulating the T cell balance both in vivo and in vitro. TDO2-overexpressed DC2.4 and TDO2-/- mouse bone marrow-derived DCs (BMDCs) were generated to verify the role of TDO2 in DC maturation and functionality. TDO2 overexpression in BMDCs via PGE2 treatment exhibited an immature phenotype and tolerogenic state, whereas TDO2-/- BMDCs exhibited a mature phenotype and a proinflammatory state. Furthermore, transplant of TDO2-overexpressed BMDCs alleviated collagen-induced arthritis severity in mice, which was correlated with a reduction in Th17 populations and an increase in regulatory T cells. Collectively, these results indicate that TDO2 plays an important role in the tolerogenic phenotype and may be a promising target for the generation tolerogenic DCs for rheumatoid arthritis treatment., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Four previously undescribed angucyclinones umezawaones A-D (1-4) were isolated from the liquid cultures of Umezawaea beigongshangensis. Their structures were determined by spectroscopic analyses, single crystal X-ray diffraction, quantum chemical 13 C NMR and electronic circular dichroism calculations. All compounds displayed strong inhibitory activities against indoleamine 2,3-dioxygenase and tryptophan-2,3-dioxygenase in enzymatic assay, especially compound 2., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2023. Published by Elsevier B.V.)

Background: In response to inflammation and other stressors, tryptophan is catalyzed by Tryptophan 2,3-Dioxygenase (TDO), which leads to activation of the kynurenine pathway. Sepsis is a serious condition in which the body responds improperly to an infection, and the brain is the inflammation target in this condition., Objective: This study aimed to determine if the induction of TDO contributes to the permeability of the Blood-Brain Barrier (BBB), mortality, neuroinflammation, oxidative stress, and mitochondrial dysfunction, besides long-term behavioral alterations in a preclinical model of sepsis., Methods: Male Wistar rats with two months of age were submitted to the sepsis model using Cecal Ligation and Perforation (CLP). The rats received allopurinol (Allo, 20 mg/kg, gavage), a TDO inhibitor, or a vehicle once a day for seven days., Results: Sepsis induction increased BBB permeability, IL-6 level, neutrophil infiltrate, nitric oxide formation, and oxidative stress, resulting in energy impairment in 24h after CLP and Allo administration restored these parameters. Regarding memory, Allo restored short-term memory impairment and decreased depressive behavior. However, no change in survival rate was verified., Conclusion: In summary, TDO inhibition effectively prevented depressive behavior and memory impairment 10 days after CLP by reducing acute BBB permeability, neuroinflammation, oxidative stress, and mitochondrial alteration., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Ethnopharmacological Relevance: The radix of Paeonia lactiflora Pall. (PaeR) is a traditional Chinese medicine (TCM) clinically used for treating depression. Although it has been established that PaeR can protect the liver and alleviate depressive-like behaviors, its bioactive chemicals and antidepressant mechanism remain unclear. Our pilot study showed that PaeR reduced the expression of the L-tryptophan- catabolizing enzyme tryptophan 2,3-dioxygenase (TDO) in the livers of stress-induced depression-like mice., Aim of the Study: This study aimed to screen potential TDO inhibitors from PaeR and investigate the potential therapeutic use of TDO inhibition for treating depression., Materials and Methods: Molecular docking, magnetic ligand fishing, and secrete-pair dual luminescence assay were conducted for in vitro ligand discovery and high-throughput screening of TDO inhibitors. Stable TDO overexpression was achieved in HepG2 cell lines to evaluate the TDO inhibitory activities of drugs in vitro by RT-PCR and Western blot analyses of TDO at mRNA and protein levels. In vivo validation of TDO inhibitory potency and evaluation of TDO inhibition as a potential therapeutic strategy for major depressive disorder (MDD) were performed using mice subjected to "3 + 1″ combined stresses for at least 30 days to induce depression-like behaviors. A well-known TDO inhibitor, LM10, was evaluated in parallel., Results: The PaeR extract significantly ameliorated depressive-like behaviors of stressed mice, attributed to inhibition of TDO expression and tryptophan modulation metabolism. After a comprehensive analysis of molecular docking, ligand fishing, and luciferase assay, paeoniflorin was screened as a TDO inhibitor from the PaeR extract. This compound, structurally different from LM10, potently inhibited human and mouse TDO in cell- and animal-based assays. The effects of TDO inhibitors on MDD symptoms were evaluated in a stress-induced depression-like mouse model. In mice, both inhibitors had beneficial effects on stress-induced depressive-like behavioral despair and unhealthy physical status. Moreover, both inhibitors increased the liver serotonin/tryptophan ratio and decreased the kynurenine/tryptophan ratio after oral administration, demonstrating in vivo inhibition of TDO activity. Our data substantiated the potential of TDO inhibition as a therapeutic strategy to improve behavioral activity and decrease despair symptoms in major depressive disorder., Conclusions: This study introduced a hitherto undocumented comprehensive screening strategy to identify TDO inhibitors in PaeR extract. Our findings also highlighted the potential of PaeR as a source of antidepressant constituents and pinpointed the inhibition of TDO as a promising therapeutic approach for managing major depressive disorder., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Molecular docking of a large set of thiosemicarbazide-based ligands resulted in obtaining compounds that inhibited both human DNA topoisomerase IIα and indoleamine-2,3-dioxygenase-1 (IDO1). To the best of our knowledge, these compounds are the first dual inhibitors targeting these two enzymes. As both of them participate in the anticancer response, the effect of the compounds on a panel of cancer cell lines was examined. Among the cell lines tested, lung cancer (A549) and melanoma (A375) cells were the most sensitive to compounds 1 (IC 50 =0.23 µg/ml), 2 (IC 50 =0.83 µg/ml) and 3 (IC 50 =0.25 µg/ml). The observed activity was even 90-fold higher than that of etoposide, with selectivity index values reaching 125. In-silico simulations showed that contact between 1-3 and human DNA topoisomerase II was maintained through aromatic moieties located at limiting edges of ligand molecules and intensive interactions of the thiosemicarbazide core with the DNA fragments present in the catalytic site of the enzyme.

Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.

Deletion of the tryptophan 2,3-dioxygenase ( TDO2 ) gene induces an anxiolytic-like behaviour in mice and TDO inhibition by allopurinol elicits an antidepressant-like effect in rats exposed to restraint stress. Chronic nicotine administration inhibits TDO activity, enhances brain serotonin synthesis and exerts anxiolytic- and antidepressant-like effects in rodent models. There is a strong association between anxiety, depression and tobacco use, which is stronger in women than in men. The present study aimed to examine the relationship between behavioural measures of anxiety and depression with liver TDO activity, brain tryptophan concentration and serotonin synthesis in rats treated chronically with nicotine. Behavioural measures included the elevated plus maze (EPM), open field (OFT) and forced swim (FST) tests. Biochemical measures included TDO activity, serum corticosterone and brain Trp, 5-HT and 5-HIAA concentrations. Anxiolytic-like and antidepressant-like effects of chronic nicotine were confirmed in association with TDO inhibition and elevation of brain Trp and 5-HT. Sex differences in behaviour were independent of the biochemical changes. At baseline, female rats performed better than males in OFT and FST. Nicotine was less anxiolytic in females in the open arm test. Nicotine treatment did not elicit different responses between sexes in the FST. Our findings support the notion that liver TDO activity exhibits a strong association with behavioural measures of anxiety and depression in experimental models, but provide little evidence for sex differences in behavioural response to nicotine. The TDO-anxiety link may be underpinned by kynurenine metabolites as well as serotonin., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygenase (TDO) catalyze the conversion of L-tryptophan to N-formyl-kynurenine and thus play primary roles in metabolism, inflammation, and tumor immune surveillance. Because their activities depend on their heme contents, which vary in biological settings and go up or down in a dynamic manner, we studied how their heme levels may be impacted by nitric oxide (NO) in mammalian cells. We utilized cells expressing TDO or IDO1 either naturally or via transfection and determined their activities, heme contents, and expression levels as a function of NO exposure. We found NO has a bimodal effect: a narrow range of low NO exposure promoted cells to allocate heme into the heme-free TDO and IDO1 populations and consequently boosted their heme contents and activities 4- to 6-fold, while beyond this range the NO exposure transitioned to have a negative impact on their heme contents and activities. NO did not alter dioxygenase protein expression levels, and its bimodal impact was observed when NO was released by a chemical donor or was generated naturally by immune-stimulated macrophage cells. NO-driven heme allocations to IDO1 and TDO required participation of a GAPDH-heme complex and for IDO1 required chaperone Hsp90 activity. Thus, cells can up- or downregulate their IDO1 and TDO activities through a bimodal control of heme allocation by NO. This mechanism has important biomedical implications and helps explain why the IDO1 and TDO activities in animals go up and down in response to immune stimulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Accumulative studies suggest that inflammatory bowel disease (IBD) may cause multiple central nervous system (CNS) pathologies. Studies have found that indoleamine-2,3-dioxygenase (IDO, rate-limiting enzyme of the kynurenine (Kyn) pathway) deficient mice were protected from endotoxin induced cognitive impairment, and Kyn administration induced cognitive memory deficits in both control and IDO-deficient mice. However, there is no investigation of the brain Kyn pathway in IBD, thus we investigated whether dextran sulfate sodium (DSS)-induced colitis could cause dysregulation of Kyn pathway in brain, and also in serum. C57BL/6J mice were given drinking water with 2% DSS for 10 consecutive days to induce colitis. In serum, we found significant increase in Kyn and kynurenic acid (Kyna) level, which was regulated by IDO-1 and KAT2 (rate-limiting enzymes of Trp-Kyn-Kyna pathway). Similarly, by analyzing GEO datasets, higher IDO-1 levels in peripheral blood monocytes and colon of UC patients was found. Furthermore, the Kyn pathway was significantly upregulated in the cerebral cortex under the action of IDO-1 after DSS treatment, which ultimately induced the neurotoxic phenotype of astrocytes. To investigate whether gut microbiota is involved in IBD-induced Kyn pathway dysregulation, we performed intestinal flora 16S rRNA sequencing and found that DSS-induced colitis significantly altered the composition and diversity of the gut microbiota. Metabolic function analysis also showed that Tryptophan metabolism, NOD-like receptor signaling pathway and MAPK signaling pathway were significantly up-regulated in the 2% DSS group. A significant association between intestinal flora and Trp metabolism (both in serum and brain) was found by correlation analysis. Overall, this study revealed that DSS-induced colitis causes dysregulation of the Kyn pathway in serum and brain by affecting rate-limiting enzymes and intestinal flora., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhao, Wu, Quan, Zhou, Hong, Niu, Li, Huang, Qiao, Zhao, Cui and Shen.)

Introduction: Tryptophan metabolism has been shown to be involved in tumor development. Two main tryptophan-degrading enzymes, tryptophan 2,3-dioxygenase (TDO2) and indoleamine 2,3-dioxygenase 1 (IDO1), may potently promote cancer cell survival and distant metastasis in diverse types of cancer, such as lung and breast cancer. IDO1 overexpression is an independent prognosticator in gastric cancer (GC). This work aimed to uncover the expression of TDO2 and its clinicopathologic significance in GC., Methods: TDO2 expression was evaluated in public data of The Cancer Genome Atlas cohort STAD and in two different GC cohorts. Correlation between TDO2 and immune cell infiltrates as well as PD-L1 tumor staining was investigated. The biofunction of TDO2 was examined with MTT, colony formation, and spheroid formation assays by RNA interference., Results: TDO2 expression was correlated with both progressive disease and clinical outcome, and its expression was an independent predictor of prognosis in GC. TDO2 expression was correlated with infiltration of immune cells and tumor expression of PD-L1. Inhibition of TDO2 expression suppressed cell proliferation, colony formation, and cell invasion of GC cells. Additionally, suppression of TDO2 expression inhibited spheroid body-formation and viability of GC organoids., Conclusion: Our data show that TDO2 might be a crucial marker for predicting prognosis and targeted therapy in GC., (© 2022 S. Karger AG, Basel.)

In the immunotherapy based on immune checkpoint inhibition (IC), additional ICs are being studied to increase its effectiveness. An almost unstudied feature is the possible co-expression of ICs, which can determine the therapeutic efficacy of their inhibition. For the selection of promising ICs, information on the association of their expression with cancer development may be essential. We have obtained data on the expression correlation of ADAM17 , PVR , TDO2 , CD274 , CD276 , CEACAM1 , IDO1 , LGALS3 , LGALS9 , and HHLA2 genes in gastric cancer (GC). All but one, TDO2 , have other IC genes with co-expression at some stage. At the metastatic stage, the expression of the IDO1 does not correlate with any other gene. The correlations are positive, but the expressions of the CD276 and CEACAM1 genes are negatively correlated. The expression of TDO2 and LGALS3 is associated with GC metastasis. The expression of TDO2 four-fold higher in metastatic tumors than in non-metastatic tumors, but LGALS3 was two-fold lower. The differentiation is associated with IDO1 . The revealed features of TDO2 , with a significant increase in expression at the metastatic stage and the absence of other IC genes with correlated expression indicates that the prospect of inhibiting TDO2 in metastatic GC. IDO1 may be considered for inhibition in low-differentiated tumors.

Background: Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) remains a global health issue. The characterized virulent M. tb H37Rv, avirulent M. tb H37Ra and BCG strains are widely used as reference strains to investigate the mechanism of TB pathogenicity. Here, we attempted to determine metabolomic signatures associated with the Mycobacterial virulence in human macrophages through comparison of metabolite profile in THP-1-derived macrophages following exposure to the M. tb H37Rv, M. tb H37Ra and BCG strains., Results: Our findings revealed remarkably changed metabolites in infected macrophages compared to uninfected macrophages. H37Rv infection specifically induced 247 differentially changed metabolites compared to H37Ra or BCG infection. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed H37Rv specifically induces tryptophan metabolism. Moreover, quantitative PCR (qPCR) results showed that indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) which converts the tryptophan to a series of biologically second metabolites were up-regulated in H37Rv-infected macrophages compared to H37Ra- or BCG-infected macrophages, confirming the result of enhanced tryptophan metabolism induced by H37Rv infection. These findings indicated that targeting tryptophan (Trp) metabolism may be a potential therapeutic strategy for pulmonary TB., Conclusions: We identified a number of differentially changed metabolites that specifically induced in H37Rv infected macrophages. These signatures may be associated with the Mycobacterial virulence in human macrophages. The present findings provide a better understanding of the host response associated with the virulence of the Mtb strain., (© 2022. The Author(s).)

Characterization of the dynamic change in the immunological landscape during malignant transformation from precancerous lesions to cancerous lesions in squamous cell carcinoma (SCC) is critical for the application of immunotherapy. Here, we performed single-cell RNA-Seq (scRNA-Seq) of 131,702 cells from 13 cancerous tissues of oral squamous cell carcinoma (OSCC), 3 samples of precancerous oral leukoplakia, and 8 adjacent normal samples. We found that tumor-infiltrating CD4+ and CD8+ T cells were functionally inhibited by immunosuppressive ligands expressed on various types of myeloid cells or neutrophils in the process of oral carcinogenesis. Notably, we identified a subset of myofibroblasts that exclusively expressed tryptophan 2,3-dioxygenase (TDO2). These TDO2+ myofibroblasts were located distally from tumor nests, and both CD4+ and CD8+ T cells were enriched around them. Functional experiments revealed that TDO2+ myofibroblasts were more likely to possess the ability for chemotaxis toward T cells but induced the transformation of CD4+ T cells into Tregs and caused CD8+ T cell dysfunction. We further showed that use of the TDO2 inhibitor LM10 attenuated the inhibitory states of T cells, restored the T cell antitumor response, and prevented the progression of OSCC malignant transformation in murine models. Our study reveals a multistep transcriptomic landscape of OSCC and demonstrates that TDO2+ myofibroblasts are potential targets for immunotherapy.

The current studies associated with tumor biology continue to describe a high correlation between tryptophan (Trp) metabolism and tumor progression. These findings reflect the complex underlying mechanism of tumor development and highlight the need to explore additional drug targets for carcinoma-associated diseases. In our study, we reported that elevated Trp metabolism was observed in highly malignant glioma tumor tissues from patients. The elevated Trp metabolism in glioma cells were induced by the overexpression of Trp 2,3-dioxygenase 2 (TDO2), which further contributed to the production of the metabolite kynurenine (Kyn). Subsequently, the Kyn derived from Trp metabolism was able to mediate the activation of the aryl hydrocarbon receptor (AhR) and downstream PI3K/AKT signals, resulting in the strengthening of tumor stemness and growth. Meanwhile, the activation of the AhR could promote the process of epithelial-mesenchymal transition in gliomas through a TGF-β-dependent mechanism, leading to enhanced tumor invasion in vitro and in vivo. Inhibition of the AhR using StemRegenin 1 was demonstrated to suppress glioma growth and improve the outcome of traditional chemotherapy in subcutaneous tumor-bearing mice, representing a promising therapeutic target for clinical glioma treatment., (© 2022 International Federation for Cell Biology.)

Background: Tryptophan 2,3-dioxygenase (TDO) encoded by TDO2 , a rate-limiting enzyme in the kynurenine pathway, catabolizes tryptophan to kynurenine, evades immune surveillance, and promotes tumor growth. Although accumulating evidence suggests a crucial role of TDO2 during tumor formation and development, systematic evaluation of TDO2 across human cancers has rarely been reported., Methods: To shed more light on the role of TDO2 in human cancer, we explored the expression profiles of TDO2 and identified its prognostic value in pancancer analysis through TCGA, CCLE, and GTEx databases. We further utilized TCGA data to evaluate the association between TDO2 and tumor immunological features, such as mismatch repair (MMR), tumor immune infiltration, immune checkpoint-related genes, tumor mutational burden (TMB), microsatellite instability (MSI), and DNA methyltransferase (DNMT)., Results: TDO2 exhibited different expression levels in various cancer cell lines. Frequently, TDO2 was detected to be highly expressed in the majority of cancers. In addition, high TDO2 expression was correlated with an unfavorable prognosis for patients in KIRP, LGG, TGCT, and UVM. Moreover, high TDO2 expression level positively correlated with higher immune infiltration, especially dendritic cells. Additionally, there is a close relationship between TDO2 and immune checkpoint-related gene markers, such as LAIR1 , CD276 , NRP1 , CD80 , and CD86 . Finally, correlation analysis has demonstrated a high-correlation between TDO2 and TMB, MSI, MMR, and DNMT of multiple cancer types., Conclusion: Therefore, our results suggest that TDO2 can function as a potential prognostic biomarker due to its role in tumor immunity regulation., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022 Jing Cui et al.)

Indoleamine 2.3-dioxygenases (IDO1/2) and tryptophan 2.3-dioxygenase (TDO) are the initial and rate-limiting enzymes in tryptophan metabolism, which play an essential role in mediating immunosuppression in tumor microenvironment. Accumulating evidence has indicated that both IDO1 and TDO are highly expressed in many malignant tumors, and their expression is generally associated with reduced tumor-infiltrating immune cells, increased regulatory T-cell infiltration, as well as cancer progression and poor prognosis for malignancies. A large number of IDO1 and TDO inhibitors have been screened or synthesized in the last two decades. Thus far, at least 12 antagonists targeting IDO1 and TDO have advanced to clinical trials. In this account, we conducted a comprehensive review of the development of IDO1 and TDO inhibitors in cancer immunotherapy, particularly their clinical research progress, and presented the current challenges and corresponding solutions., Competing Interests: Hao Yang is affiliated with POWERCHINA Chengdu Engineering Corporation Limited, Chengdu, 610072, Sichuan, China. The authors report no other conflicts of interest in this work., (© 2022 Peng et al.)

Indoleamine 2, 3-dioxygenase 1 (IDO1) is a rate-limiting enzyme that catalyzes the kynurenine (Kyn) pathway of tryptophan metabolism in the first step, and the kynurenine pathway plays a fundamental role in immunosuppression in the tumor microenvironment. Therefore, researchers are vigorously developing IDO1 inhibitors, hoping to apply them to cancer immunotherapy. Nowadays, there have been 11 kinds of IDO1 inhibitors entering clinical trials, among which many inhibitors have shown good tumor inhibitory effect in phase I/II clinical trials. But the phase III study of the most promising IDO1 inhibitor compound 29 (Epacadostat) failed in 2018, which may be caused by the compensation effect offered by tryptophan 2,3-dioxygenase (TDO), the mismatched drug combination strategies, or other reasons. Luckily, dual-target inhibitors show great potential and advantages in solving these problems. In recent years, many studies have linked IDO1 to popular targets and selected many IDO1 dual-target inhibitors through pharmacophore fusion strategy and library construction, which enhance the tumor inhibitory effect and reduce side effects. Currently, three kinds of IDO1/TDO dual-target inhibitors have entered clinical trials, and extensive studies have been developing on IDO1 dual-target inhibitors. In this review, we summarize the IDO1 dual-target inhibitors developed in recent years and focus on the structure optimization process, structure-activity relationship, and the efficacy of in vitro and in vivo experiments, shedding a light on the pivotal significance of IDO1 dual-target inhibitors in the treatment of cancer, providing inspiration for the development of new IDO1 dual-target inhibitors., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Inactivation of adenomatous polyposis coli (APC) is common across many cancer types and serves as a critical initiating event in most sporadic colorectal cancers. APC deficiency activates WNT signaling, which remains an elusive target for cancer therapy, prompting us to apply the synthetic essentiality framework to identify druggable vulnerabilities for APC-deficient cancers. Tryptophan 2,3-dioxygenase 2 (TDO2) was identified as a synthetic essential effector of APC-deficient colorectal cancer. Mechanistically, APC deficiency results in the TCF4/β-catenin-mediated upregulation of TDO2 gene transcription. TDO2 in turn activates the Kyn-AhR pathway, which increases glycolysis to drive anabolic cancer cell growth and CXCL5 secretion to recruit macrophages into the tumor microenvironment. Therapeutically, APC-deficient colorectal cancer models were susceptible to TDO2 depletion or pharmacologic inhibition, which impaired cancer cell proliferation and enhanced antitumor immune profiles. Thus, APC deficiency activates a TCF4-TDO2-AhR-CXCL5 circuit that affects multiple cancer hallmarks via autonomous and nonautonomous mechanisms and illuminates a genotype-specific vulnerability in colorectal cancer., Significance: This study identifies critical effectors in the maintenance of APC-deficient colorectal cancer and demonstrates the relationship between APC/WNT pathway and kynurenine pathway signaling. It further determines the tumor-associated macrophage biology in APC-deficient colorectal cancer, informing genotype-specific therapeutic targets and the use of TDO2 inhibitors. This article is highlighted in the In This Issue feature, p. 1599., (©2022 American Association for Cancer Research.)

Background: Tryptophan 2,3-dioxygenase (TDO2) is the primary enzyme that catabolizes tryptophan to kynurenine. Numerous studies have suggested that TDO2 is involved in inflammation-related diseases. However, its role in osteoarthritis (OA) has not yet been investigated. The aim of the present study was to explore the levels of TDO2 in the synovium and synovial fluid (SF) of patients with OA and its correlation with clinical manifestations and levels of pro-inflammatory cytokines.  METHODS : Synovium and SF samples were collected from patients with OA and patients with joint trauma (controls) during surgery. An enzyme-linked immunosorbent assay (ELISA) was used to measure TDO2, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) levels in the synovium and SF. Diagnostic performance of TDO2 in the synovium to discriminate between controls and OA patients was assessed using receiver operating characteristic (ROC) curve analysis. Correlations between TDO2 levels, OA clinical features, and pro-inflammatory cytokines were evaluated using Pearson correlation analysis. Effects of IL-1β or TNF-α stimulation on TDO2 expression in OA-fibroblast-like synoviocytes (OA-FLS) were also examined., Results: The levels of TDO2, IL-1β, and TNF-α in the synovium of patients with OA were found to be significantly higher than those in controls. ROC curve analysis revealed an area under the curve (AUC) of 0.800 with 64.3% sensitivity and 85.0% specificity of TOD2 in the synovium, which enabled discriminating patients with OA from controls. Moreover, protein expression of TDO2 was upregulated to a greater extent in OA-FLS than in normal synovial fibroblasts (NSF). Furthermore, the levels of TDO2 showed significantly positive correlation with IL-1β and TNF-α levels in the synovium and SF. TDO2 levels in the synovium were also positively correlated with the Kellgren-Lawrence score. Additionally, TDO2 protein expression was significantly increased in IL-1β‒ or TNF-α‒stimulated OA-FLS than in control FLS., Conclusion: These data indicate that highTDO2 levels in the synovium can be correlated with pro-inflammatory cytokines and severity of OA., (© 2022. The Author(s).)

Aberrant amino acid metabolism is a common event in obesity. Particularly, subjects with obesity are characterized by the excessive plasma kynurenine (Kyn). However, the primary source of Kyn and its impact on metabolic syndrome are yet to be fully addressed. Herein, we show that the overexpressed indoleamine 2,3-dioxygenase 1 (IDO1) in adipocytes predominantly contributes to the excessive Kyn, indicating a central role of adipocytes in Kyn metabolism. Depletion of Ido1 in adipocytes abrogates Kyn accumulation, protecting mice against obesity. Mechanistically, Kyn impairs lipid homeostasis in adipocytes via activating the aryl hydrocarbon receptor (AhR)/Signal transducer and activator of transcription 3 /interleukin-6 signaling. Genetic ablation of AhR in adipocytes abolishes the effect of Kyn. Moreover, supplementation of vitamin B6 ameliorated Kyn accumulation, protecting mice from obesity. Collectively, our data support that adipocytes are the primary source of increased circulating Kyn, while elimination of accumulated Kyn could be a viable strategy against obesity., (© 2022. The Author(s).)

Background and Purpose: Abnormal kynurenine (Kyn) metabolism has been closely linked to the pathogenesis of rheumatoid arthritis (RA). The aims of this study were to investigate the role of tryptophan 2,3-dioxygenase 2 (TDO2), a rate-limiting enzyme that converts tryptophan (Trp) to Kyn, in regulating fibroblast-like synoviocyte (FLS)-mediated synovial inflammation in autoimmune arthritis., Experimental Approach: The expression of TDO2 was determined by immunohistochemistry, confocal laser scanning fluorescence microscopy, imaging flow cytometry and Western blot. TDO2 activity was tested by HPLC and colorimetric assay. TDO2 siRNA and TDO2 inhibitor 680C91 were used to inhibit TDO2 in AA-FLS function in vitro. A rat model of adjuvant-induced arthritis (AA) was used to evaluate the in vivo effect of allopurinol (Allo), a TDO2 inhibitor., Key Results: TDO2 expression was strongly increased in synovial tissue and FLS of RA and AA. Immune cells were found to express high amount of TDO2 proteins at the peak stage of AA. Pharmacological inhibition or knockdown of TDO2 in AA-FLS resulted in a reduced proliferation, secretion, migration and invasion. Kyn restored the inhibitory effect of TDO2 inhibition on activation of AA-FLS. Allo treatment ameliorated the arthritis severity and decreased the activity of TDO2., Conclusion and Implications: Our results suggest that elevated TDO2 expression may contribute to synovial inflammation and joint destruction during arthritis. Therefore, targeting TDO2 activity and the Kyn pathway of Trp degradation may represent a potential therapeutic strategy in RA., (© 2021 The British Pharmacological Society.)

Liver cancer is a malignant cancer phenotype for which there currently remains a lack of reliable biomarkers and therapeutic targets for disease management. Tryptophan 2,3‑dioxygenase (TDO2), a heme‑containing polyoxygenase enzyme, is primarily expressed in cells of the liver and nervous systems. In the present study, through the combination of cancer bioinformatics and analysis of clinical patient samples, it was shown that TDO2 expression in liver cancer tissue samples was significantly higher than that in normal tissues, and liver cancer patients with high TDO2 expression had a poor prognosis. Mechanistic studies on liver cancer cells showed that TDO2 promoted cancer cell migration and invasion via signal transduction through the Wnt5a pathway. Such regulation impacted the expression of cancer‑associated biomarkers, such as matrix metalloprotease 7 (MMP7) and the cell adhesion receptor CD44. Treatment with a calcium channel blocker (azelnidipine) reduced TDO2 levels and inhibited liver cancer cell migration and invasion. A mouse xenograft cancer model showed that TDO2 promoted tumorigenesis. Furthermore, azelnidipine treatment to downregulate TDO2 also decreased liver cancer development in this mouse cancer model. TDO2 is thus not only a useful liver cancer biomarker but a potential drug target for management of liver cancer.

Tumors are heterogeneous cellular environments with entwined metabolic dependencies. Here, we use a tumor transcriptome deconvolution approach to profile the metabolic states of cancer and non-cancer (stromal) cells in bulk tumors of 20 solid tumor types. We identify metabolic genes and processes recurrently altered in cancer cells across tumor types, highlighting pan-cancer upregulation of deoxythymidine triphosphate (dTTP) production. In contrast, the tryptophan catabolism rate-limiting enzymes IDO1 and TDO2 are highly overexpressed in stroma, raising the hypothesis that kynurenine-mediated suppression of antitumor immunity may be predominantly constrained by the stroma. Oxidative phosphorylation is the most upregulated metabolic process in cancer cells compared to both stromal cells and a large atlas of cancer cell lines, suggesting that the Warburg effect may be less pronounced in cancer cells in vivo. Overall, our analysis highlights fundamental differences in metabolic states of cancer and stromal cells inside tumors and establishes a pan-cancer resource to interrogate tumor metabolism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Prostate cancer (PC) is the second leading cause of death in men in the US. PC has a high recurrence rate, and limited therapeutic options are available to prevent disease recurrence. The tryptophan-degrading enzymes 2,3-indoleamine dioxygenase (IDO1) and tryptophan dioxygenase (TDO2) are upregulated in invasive PC. (1 S ,2 E ,4 R ,6 R ,7 E ,11 E )-2,7,11-cembratriene-4,6-diol (β-CBT) and its C-4 epimer α-CBT are the precursors to key flavor ingredients in tobacco leaves. Nearly 40-60% of β- and α-CBT are purposely degraded during commercial tobacco fermentation. Earlier, β-CBT inhibited invasion, reversed calcitonin-stimulated transepithelial resistance decrease, and induced tighter intercellular barriers in PC-3M cells. This study demonstrates the in vitro β-CBT anti-migratory (wound-healing assay) and anti-clonogenicity (colony-formation assay) activities against five diverse human PC cell lines, including the androgen-independent PC-3, PC-3M, and DU-145, the castration-recurrent CWR-R1ca, and the androgen-dependent CWR-22rv1. Meanwhile, β-CBT potently suppressed in vivo locoregional and distant recurrences after the primary tumor surgical excision of PC-3M-Luc cell tumor engrafted in male nude mice. β-CBT treatments suppressed organ and bone metastasis and lacked any major toxicity over the 60-day study course. β-CBT treatments significantly suppressed IDO1, TDO2, and their final metabolite kynurenine levels in PC-3M cells. β-CBT treatments significantly suppressed the tumor recurrence marker PSA and kynurenine levels in treated animals' plasma. β-CBT emerges as a promising PC recurrence suppressive lead.

Uterine leiomyomas (fibroids) are common benign tumors in women. The tryptophan metabolism through the kynurenine pathway plays important roles in tumorigenesis in general. Leiomyomas expressing mutated mediator complex subunit 12 (mut-MED12) were reported to contain significantly decreased tryptophan levels; the underlying mechanism and the role of the tryptophan metabolism-kynurenine pathway in leiomyoma tumorigenesis, however, remain unknown. We here assessed the expression and regulation of the key enzymes that metabolize tryptophan. Among these, the tissue mRNA levels of tryptophan 2,3-dioxygenase (TDO2), the rate limiting enzyme of tryptophan metabolism through the kynurenine pathway, was 36-fold higher in mut-MED12 compared to adjacent myometrium (P < 0.0001), and 14-fold higher compared to wild type (wt)-MED12 leiomyoma (P < 0.05). The mRNA levels of other tryptophan metabolizing enzymes, IDO1 and IDO2, were low and not significantly different, suggesting that TDO2 is the key enzyme responsible for reduced tryptophan levels in mut-MED12 leiomyoma. R5020 and medroxyprogesterone acetate (MPA), two progesterone agonists, regulated TDO2 gene expression in primary myometrial and leiomyoma cells expressing wt-MED12; however, this effect was absent or blunted in leiomyoma cells expressing G44D mut-MED12. These data suggest that MED12 mutation may alter progesterone-mediated TDO2 expression in leiomyoma, leading to lower levels of tryptophan in mut-MED12 leiomyoma. This highlights that fibroids can vary widely in their response to progesterone as a result of mutation status and provides some insight for understanding the effect of tryptophan-kynurenine pathway on leiomyoma tumorigenesis and identifying targeted interventions for fibroids based on their distinct molecular signatures., (© 2022. The Author(s).)

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme 1-4 . Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides 'substitutants' to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity., (© 2022. The Author(s).)

Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygense (TDO) are heme-containing dioxygenases that catalyze the conversion of tryptophan to N-formyl-kynurenine and thus enable generation of l-kynurenine and related metabolites that govern the immune response and broadly impact human biology. Given that TDO and IDO1 activities are directly proportional to their heme contents, it is important to understand their heme delivery and insertion processes. Early studies established that TDO and IDO1 heme levels are sub-saturating in vivo and subject to change but did not identify the cellular mechanisms that provide their heme or enable dynamic changes in their heme contents. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into IDO1 and TDO expressed in both normal and heme-deficient HEK293T cells and into IDO1 naturally expressed in HeLa cells in response to IFN-γ, and also investigated the interactions of TDO and IDO1 with GAPDH and Hsp90 in cells and among their purified forms. We found that GAPDH delivered both mitochondrially-generated and exogenous heme to apo-IDO1 and apo-TDO in cells, potentially through a direct interaction with either enzyme. In contrast, we found Hsp90 interacted with apo-IDO1 but not with apo-TDO, and was only needed to drive heme insertion into apo-IDO1. By uncovering the cellular processes that allocate heme to IDO1 and TDO, our study provides new insight on how their activities and l-kynurenine production may be controlled in health and disease., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Metabolism of tryptophan (Trp), an essential amino acid, represent a major metabolic pathway that both promotes tumor cell intrinsic malignant properties as well as restricts antitumour immunity, thus emerging as a drug development target for cancer immunotherapy. Three cytosolic enzymes, namely indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan 2,3-dioxygenase (TDO2), catalyzes the first-rate limiting step of the degradation of Trp to kynurenine (Kyn) and modulates immunity toward immunosuppression mainly through the aryl hydrocarbon receptor (AhR) activation in numerous types of cancer. By restoring antitumor immune responses and synergizing with other immunotherapies, the encouraging preclinical data of IDO1 inhibitors has dramatically failed to translate into clinical success when combined with immune checkpoints inhibitors, reigniting the debate of combinatorial approach. In this review, we i) provide comprehensive evidences on immunomodulatory role of the Trp catabolism metabolites that highlight this pathway as relevant target in immuno-oncology, ii)ii) discuss underwhelming results from clinical trials investigating efficacy of IDO1 inhibitors and underlying mechanisms that might have contributed to this failure, and finally, iii) discuss the current state-of-art surrounding alternative approaches of innovative antitumor immunotherapies that target molecules of Trp catabolism as well as challenges and perspectives in the era of immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Peyraud, Guegan, Bodet, Cousin, Bessede and Italiano.)

Selenium is an underexplored element that can be used for bioisosteric replacement of lower molecular weight chalcogens such as oxygen and sulfur. More studies regarding the impact of selenium substitution in different chemical scaffolds are needed to fully grasp this element's potential. Herein, we decided to evaluate the impact of selenium incorporation in a series of tryptophan 2,3-dioxygenase (TDO2) inhibitors, a target of interest in cancer immunotherapy. First, we synthesized the different chalcogen isosteres through Suzuki-Miyaura type coupling. Next, we evaluated the isosteres' affinity and selectivity for TDO2, as well as their lipophilicity, microsomal stability and cellular toxicity on TDO2-expressing cell lines. Overall, chalcogen isosteric replacements did not disturb the on-target activity but allowed for a modulation of the compounds' lipophilicity, toxicity and stability profiles. The present work contributes to our understanding of oxygen/sulfur/selenium isostery towards increasing structural options in medicinal chemistry for the development of novel and distinctive drug candidates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Masson SAS.)

Competing Interests: LB has participated in an advisory board to Incyte Inc. on epacadostat in melanoma and was invited to give a MSL seminar to Incyte Inc. on IDO (2017). GP declares a conflict of interest due to his role as a co-inventor in the discovery and development of IDO/TDO inhibitor technologies patented by the Lankenau Institute for Medical Research and licensed to Duet Therapeutics, Inc., a private company for which he serves presently as a scientific advisor. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Tryptophan 2,3-dioxygenase (TDO2) was an initial rate-limiting enzyme of the kynurenine (Kyn) pathway in tryptophan (Trp) metabolism. We undertook this study to determine a comprehensive analysis of TDO2 expression in immune cells and assess the characterization of immune cell phenotype in TDO2 knockout mice. The expression of TDO2 in various tissues of DBA/1 mice was detected by quantitative real-time PCR (qPCR) and immunohistochemistry. Both flow cytometry and immunofluorescence were used to analyze the expression of TDO2 in immune cells. Furthermore, TDO2 knockout (KO) mice were generated by CRISPR/Cas9 technology to detect immune cell phenotype. TDO2 protein level in liver was tested by western blot. High-performance liquid chromatography was used to detect the level of Trp and Kyn. Flow cytometry was used to test the proportions of splenic lymphocyte subsets in wild-type (WT) and TDO2 KO mice. We found that TDO2 was expressed in various tissues and immune cells, and TDO2 staining was mainly observed in the cytoplasm of cells. There was no difference in the development of immune cells between TDO2 KO mice and WT mice, including T cells, B cells, memory B cells, plasma cells, dendritic cells, and natural killer cells. Interestingly, the reduced M1/M2 ratio was observed in the peritoneal macrophages of TDO2 KO mice. Taken together, these findings enriched the known expression profile of TDO2, especially its expression in immune cells. Our study suggested that TDO2-mediated Trp-Kyn metabolism pathway might be involved in the immune response., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Purpose: Induction of IDO depends on the activation of AhR forming the AhR/IDO axis. Activated AhR can transcribe various target genes including cytotoxic and inhibiting receptors of NK cells. We investigated whether AhR and IDO levels as well as activating (NKG2D) and inhibiting (KIR2DL1) NK cell receptors are influenced by acute exercise and different chronic endurance exercise programs., Methods: 21 adult breast and prostate cancer patients of the TOP study (NCT02883699) were randomized to intervention programs of 12 weeks of (1) endurance standard training or (2) endurance polarized training after a cardiopulmonary exercise test (CPET). Serum was collected pre-CPET, immediately post-CPET, 1 h post-CPET and after 12 weeks post-intervention. Flow cytometry analysis was performed on autologous serum incubated NK-92 cells for: AhR, IDO, KIR2DL1 and NKG2D. Differences were investigated using analysis-of-variance for acute and analysis-of-covariance for chronic effects., Results: Acute exercise: IDO levels changed over time with a significant increase from post-CPET to 1 h post-CPET (p = 0.03). KIR2DL1 levels significantly decreased over time (p < 0.01). NKG2D levels remained constant (p = 0.31). Chronic exercise: for both IDO and NKG2D a significant group × time interaction, a significant time effect and a significant difference after 12 weeks of intervention were observed (IDO: all p < 0.01, NKG2D: all p > 0.05)., Conclusion: Both acute and chronic endurance training may regulate NK cell function via the AhR/IDO axis. This is clinically relevant, as exercise emerges to be a key player in immune regulation., (© 2021. The Author(s).)

The kynurenine pathway is the major route of tryptophan metabolism. The first step of this pathway is catalysed by one of two heme-dependent dioxygenase enzymes - tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) - leading initially to the formation of N-formylkynurenine (NFK). In this paper, we present a crystal structure of a bacterial TDO from X. campestris in complex with l-kynurenine, the hydrolysed product of NFK. l-kynurenine is bound at the active site in a similar location to the substrate (l-Trp). Hydrogen bonding interactions with Arg117 and the heme 7-propionate anchor the l-kynurenine molecule into the pocket. A mechanism for the hydrolysis of NFK in the active site is presented., (Copyright © 2021. Published by Elsevier Inc.)

Background: Bladder cancer (BC) is the 10th most common cancer in the world. BC with muscle invasion results in a poor prognosis and is usually fatal. Cancer cell metabolism has an essential role in the development and progression of tumors. Expression of tryptophan 2,3-dioxygenase (TDO2) is associated with tumor progression and worse survival in some other cancers. However, no studies have been performed to uncover the biofunctional roles of TDO2 in BC., Aim: This study aim to investigate the clinicopathologic significance of TDO2 in BC., Methods and Results: TDO2 expression was evaluated by qRT-PCR and immunohistochemistry in an integrated analysis with the Cancer Genome Atlas (TCGA) and other published datasets. TDO2 overexpression was significantly associated with T classification, N classification, and M classification, tumor stage, recurrence, and basal type, and with the expression of CD44 and aldehyde dehydrogenase 1 (ALDH1) in BC. High TDO2 expression correlated with poor outcome of BC patients. Using BC cell lines with knockdown and forced expression of TDO2, we found that TDO2 was involved in the growth, migration, and invasiveness of BC cells. Moreover, TDO2 was found to be crucial for spheroid formation in BC cells. Importantly, TDO2 promoted BC cells resistance to cetuximab through integration of the EGFR pathway., Conclusion: Our results indicate that TDO2 might take an essential part in BC progression and could be a potential marker for targeted therapy in BC., (© 2021 The Authors. Cancer Reports published by Wiley Periodicals LLC.)

A tryptophan-2,3-dioxygenase 2 (TDO2)-targeted Pt(IV) prodrug, DN604-TDOi, was designed to prove that the multi-action compound could overcome drug resistance and relieve immunosuppression via introducing a TDO2 inhibitor to the axial position of a six-coordinate Pt(IV) hybrid. Several in vitro biological studies on cisplatin-resistant NSCLC cancer cells suggested that TDO2-targeted Pt(IV) prodrug could combat cisplatin resistance via influencing TDO2-kynurenine (Kyn)-aryl hydrocarbon receptor (AhR)-Aquaporin-4 (AQP4) metabolic circuity and AhR-human DNA polymerase (hpol) κ-induced translesion DNA synthesis (TLS) genomic instability, which are positive in drug-resistant human tumors associated with malignant progression and poor survival. Remarkably, we observed that DN604-TDOi could inhibit TDO2-mediated constitutive Kyn-AhR-AQP4 signaling pathway and suppress hpol κ expression, leading to potential decrease of cell motility and genomic instability in A549/cDDP cells. It was confirmed that TDO2-targeted Pt(IV) prodrug could harness Kyn-AhR-AQP4 metabolic circuitry and TLS genomic instability, exerting antitumor effects in C57BL6 but not TDO2 -/- mice. Moreover, the Pt(IV) prodrug improved the intratumoral infiltration of T eff cells and reduced the recruitment of T reg cells. The results provided compelling preclinical evidence that TDO2-targeted Pt(IV) prodrug could abrogate immune chemotherapeutic resistance via decaying TDO2-mediated Kyn-AhR-AQP4 immunosuppression and AhR-hpol κ-induced TLS genomic instability, underscoring the development of a novel Pt(IV)-based candidate as a potent immunotherapeutic agent for chemo-immune resistance prevention., (Copyright © 2021 Elsevier Inc. All rights reserved.)